THE FIXATION BETWEEN BIOCHEMICAL AND MOFPHOLOGICAL DIFFERENTIATION IN BLASTOCLADIELLA EMERSONII By James S . Lovett A THESIS Submitted to the School fo r Advanced Graduate S tu d ies of Michigan S ta te U n iv e r sity o f A g ricu ltu re and Applied S cien ce in p a r tia l fu lf illm e n t of th e requirem ents fo r the degree of DOCTOR OF PHILOSOPHY Department o f Botany and Plant Pathology 19^9 ProQuest Number: 10008631 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008631 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 3U l l f H -'i-ic ACKNOWLEDGMENT The author w ishes to express h is s in c e r e a p p re c ia tio n to Dr. E. C. Cantino fo r h is e n th u s ia s tic support and ab le guidance during th e course o f t h is stu d y . To Ha?, e l iv THE RELATION BETWEEN BIOCHEMICAL AND MORPHOLOGICAL DIFFERENTIATION IN BLASTOCLADIELLA EMERSONII By James S . Lovett AN ABSTRACT Submitted to th e School fo r Advanced Graduate S tu d ies o f Michigan S ta te U n iv e r sity o f A g ricu ltu re and Applied S cien ce in p a r tia l fu lf illm e n t o f the requirem ents fo r the degree of DOCTOR. OF PHILOSOPHY Department o f Botany and Plant Pathology 1959 Approved ABSTRACT The enzyme glucosam ine sy n th eta se i, glut am ine-17- 6-P transam idase) was p u r ifie d c a . 1 9 -fo ld from e x tr a c ts o f the a q u atic Phycomycete B la s t o c la d ie lla e m e r so n ii, by c en tr ifu g a tio n ,, protamine s u lf a t e , f r a c t io n a t io n , and adsorption on tr.icalc.ium phosphate g e l. The pH optimum, th e tim e c o u rse, and r e la t io n between enzyme co n cen tra tio n and r e a c tio n r a te were e sta b lis h e d fo r th e p a r t ia lly p u r ifie d sy n th e ta s e . The r e a c tio n c a ta ly se d by the enzyme is o la t e d from B la st o c la d ie lla was found to be the same as th at o f the enzyme o f Neurospora c r a ssa j i . e . , d -fr u c t os e - 6- phosphate 4- 1 -g lu ta m in e — *>►d -g lu e os amine-6 - phosphate + 1-g.lutam ic a c id . In prelim in ary s tu d ie s concerning the b iochem ical b a sis o f d i f ­ f e r e n t ia t io n in B la s t .o c la d ie lla , th e fr e e amino acid pools were e x tr a cte d and compared between th e two a lte r n a tiv e mature forms o f the organism, th e z o o sp o ra n g ia l, and r e s is t a n t sp o ra n g ia l p lan ts . The l a t t e r were found to c o n ta in only 50 % as much so lu b le amino acid n itro g en as th e former, and th ere were, as w e ll, c e r ta in q u a lita tiv e d iffe r e n c e s between the two ty p e s . En a d d itio n the a c t iv it y of glucosamine synth e­ ta s e in zoosp ores, mature zoosp oran gial p la n ts , and mature r e s is ta n t sp oran gial p la n ts was e s ta b lis h e d . The unexplained d iffe r e n c e s among the variou s forms o f th e organism made a more r efin e d experim ental approach to the problem of d if f e r e n t i a ­ tio n a n e c e s s it y . T herefore, a procedure was developed fo r growing w e ll synchronized , la r g e s c a le c u ltu r e s o f r e s is ta n t sp o ra n g ia l p lan ts vi o f B la st o c la d ie lla throughout the. complete gen era tio n period in a g lu c o se-p e p to n e-y ea st medium c o n ta in in g b ica rb o n a te. The in c r e a se s in s i c e and dry weight o f in d iv id u a l p lan ts were determ ined, and a photom icrographic record of t h e ir developm ental morphology o b ta in ed , during th e growth o f c u ltu re s under such c o n d itio n s . In s tu d ie s of the g e n e sis of the r e s is ta n t sporangium, th e a c t i v i t y o f th e sy n th e ta s e , glu cose-6-p h osp h ate dehydrogenase, and phosphoglucose isom erase enzymes was determined during development in synchronous c u lt u r e . U t ili z in g the same c u ltu rin g te c h n iq u e s, th e time sequence of c h it i n , l i p i d , m elanin, and n itro g en s y n th e sis was e s t a b lis h e d . The fr e e n itro g e n pools in d evelop in g r e s is t ant s p o r a n g ia l' p la n ts were shown to undergo both q u a n tita tiv e and q u a lita tiv e changes during d iffe r e n tia tio n . Using th e approach o f comparative bioch em istry th e s ig n ific a n c e o f the changes in the c e llu la r components as th ey r e la te d to the s tr u c tu r e and fu n c tio n o f th e develop in g r e s is ta n t sp oran gia! plant was d is c u s s e d . An attempt was made to in te g r a te the p h y s io lo g ic a l and m orphological p rocesses in volved in the i n i t i a t i o n and d if f e r e n t ia t io n o f th e in d iv id u a l r e s is ta n t sporangium o f B l a s t o c l a d i e l l a . v ii 9 TABLE OF CONTENTS Page INTRODUCTION........................ 1 LITERATURE REVIEW.......................................................................................................... U Morphogenetic S tu d ies o f B la s t o c l a d ie lla e m e r so n ii. — . . . . . . . . . Glucosamine S yn th etase and th e B io sy n th e sis o f C h i t i n . . . . . . . . . . U II MATERIALS AHD METHODS.............................. C u ltu rin g and H arvestin g P roced ures............................................................ A n a ly tic a l Methods ........................................... Preparat iv e Procedures .................................................................... Sources of Chemicals and B io c h e m ic a ls ......... EXPERIMENTAL ................................................................................... The Free Amino Acid Pools in B la st o c l a d i e l l a ................................... Glucosamine S yn th etase in Plant E x tra cts ......... ^......................... The Product o f Glucosamine S yn th etase A c t i v i t y . . . ................... P u r ific a tio n o f Glucosamine S y n th e ta s e ........... P u r ific a tio n P roced ures..................... The pH Optimum o f Glucosamine S y n th e ta se ................... Time Course o f the Glucosamine Syn th etase R e a c t i o n . . . . Glucosamine Production v s . Enzyme C on cen tration ................. S u bstrate S p e c if i c it y ................. S toich iom etry o f the Glucosamine S yn th etase R ea ctio n ................ S tu d ies of R. S . Ontogeny..................................... Glucosamine S yn th etase A o tiv ity in Z oospores, O.C. P la n ts, and P .S . P lants .......................... C ultural C onditions fo r Synchronized Growth. ........... Growth S tu d ies w ith Synchronized C u ltu r es Glucosamine S yn th etase A c tiv ity During R.. S . D e v e lo p m e n t.... G lu cose-6 - phosphate Dehydrogenase and Isomerase A c tiv ity During R..S . Developm ent........................ C h itin Formation in D eveloping R..S ........................ Melano genes i s During R .S . Ontogeny ................... L ipid S y n th esis During R.S . Developm ent ........... N itrogen Transform ation in D eveloping R..S . P lants . . . . . . . . . . . v iii 18 18 22 30 32 3i 3137 39 h2 h3 hS U6 U6 U6 £l 5? 58 60 63 68 7U 80 81 81 82 TABLE OF CONTENTS - Continued Page DISCUSSION....................................................................... ■................................................. 89 The Glucosam ine-6 - phosphate S y n th e sizin g Enzyme in 89 - B .la s t o c la d ie lla ................................................................................................. Growth vs . D if f e r e n t ia t io n in B ia s to c la d ie 1 1 a .......... . ................. 90 90 The P oin t of No R eturn........................................................... O ntogenetic Changes on a Per Plant B a s i s ............................................ 91 N itrogen Metabolism and D if f e r e n t ia t i o n .............................................. 97 General C o n clu sio n s........................................................................................... 101 SUMMARY................................................................................................................................ 107 LtST OF REFERENCES....................................................................................................... 10 8 APPENDICES............................................................................................................................ 116 I L is t o f A bbreviations .............................. 116 I I Two-Dimensional Chromatographic Map of Amino A c i d s . . . . . . ............117 III Chromatographic M o b ilitie s o f S e le c te d Compounds..................... 119 IV' Summary o f Attempts to P u rify Glucosamine S y n th e ta s e ..................... 120 ix LIST OF TABLES TABLE I Page A n alysis o f the S o lu b le Amino Acid Pools in O.C. end R . S . P lan ts of B i a s t o c l a d i e l l a ....................... -................................................... 36 Gel E lu tio n w ith H exose-p hosphates............................................. k5 III Glucosamine S yn th etase P u r i f i c a t i o n . . . .............................................. 50 ■IV C ontrol R eaction M ixtures I n a c tiv e w ith the P a r t ia lly P u r ifie d Enzyme .......................................................................... $0 S to ich io m etry o f the Glucosamine S yn th etase R e a c t i o n . . . 52 H exose-phosphate Conversion During Incubation w ith the P a r t ia lly P u rifie d Glucosamine S y n th e ta s e . .......... 53 G lucose-6-phosphate Dehydrogenase Assay fo r Isomerase A c t iv it y in the P a r t ia lly P u rifie d Enzyme........................................ 5U Comparison o f the E ffic ie n c y o f G lu co se-6 -phosphate and F ru ctose-6-ph osp hate as S u b stra tes fo r the Glucosamine S yn th etase R eaction During P u r ific a tio n . ........... 56 IE V VE VIE VIII IX X XI XII XEIT XIV Glucosamine S yn thetase A c t iv it y in Z oosp ores................................ 58 Glucosamine S yn th etase A c tiv ity in Mature R .S .............. 59 A Comparison o f Glucosamine S yn thetase A c tiv ity in Z oospores, O.C. P la n ts , and R. S . P la n ts .......................................... 59 S iz e Measurements o f D eveloping R . S ............................................... 6? Dry W eights o f P lan ts at D iffe r e n t Ages During R.S. Development ...................... 69 G lu cose-6 - phosphate Dehydrogenase A c tiv ity During Develop­ ment o f R .S . P la n t s ........................................................... 75 XV The R e la tiv e A c t iv it ie s o f F ructose-6-ph osp hate and G lucose6-phosphate in the Glucosamine S yn th etase and G lu cose-6 - phos­ phate Dehydrogenase Enzyme Systems During R.S. D evelopm ent.. 16 XVI The C h itin Content o f R.S. P lants Grown in Media w ith D iffe r e n t Sodium B icarbonate C o n c e n tr a tio n s ................................. x 81 LIST OF ILLUSTRATIONS Figure Page 1 . Sugar-phosphate and Hexosamine-phosphate I n te r c o n v e r s io n s .. 17 2 . The S o lu b le Amino Acid Pools o f O.C. and R. S. P l a n t s . . . . . . . 35 3 . G lucosam ine- 6 -phosphate P u r ific a tio n Procedure . . . . . . . . . . . . . UO I4 . Glucosamine S yn th etase P u r ific a tio n P r o c e d u r e ................... . U8 5- The R e la tio n Between Glucosamine S yn th etase A c tiv ity and pH I4.9 6 . Time Course o f th e Glucosamine Syn th etase R e a ctio n ............ Ii9 7 . The E ffe c t o f Glucosamine S yn th etase C oncentration on th e R eaction P a te ............................................................................... 8 . A Thick-W alled R. S. Plant Produced in Super-optim al B icarbonate C o n cen tra tio n s................................................................ U9 62 9 . Photomicrographs o f R.S. P lants During Development in Synchronous C u ltu re............................................................................... 6 I4-66 10A-B. The Volume o f an R. S, Plant During Development ............. 11A-B. The Dry Weight of an R.S. Plant During D e v elo p m e n t......... 71 7-1 1 2 . The S p e c if ic A c tiv ity of Glucosamine S yn th etase in R.S. P lan ts During Developm ent........................................... 72 13 • T otal Glucosamine S yn th etase Per Unit Weight o f Organism During R .S . Developm ent..................................................................... 72 lltA-B . Glucosamine S yn th etase A c tiv ity Per Plant During R.S. Development ....................... 1 5 . T otal Glucosamine S yn th etase Per Unit Weight o f Organism During R.S. Development in Super-optim al Bicarbonate C o n cen tra tio n s........................................... 72 78 1 6 . The S p e c if ic A c tiv ity o f G lu cose- 6 -phosphate Dehydrogenase in R .S . P lan ts During Developm ent................................................. 78 17A-B. G lu cose- 6 - phosphate Dehydrogenase A c tiv ity Per Plant During R .S . Development ............... X I 79 LIST OF ILLUSTRATIONS - Continued Figure Page 1 8 . The C h itin Content Per Unit Weight o f Organism During R.S.'" D evelopm ent........................................................................................................ 79 19* The C h itin Content Per P lant During R.S. D e v e l o p m e n t . . . . . . . 79 20. The Melanin Content Per Unit Weight o f Organism During R.S. D evelopm ent.......................................................................................... 79 2.1. The Melanin Content Per Plant During R. S. Developm ent 79 2 2 . The L ipid Content Per Unit Weight o f Organism During R.S. Developm ent............................................................................................ 8U 2 3 . The Lipid ContentPer Plant During R .S . Development . . . . . . . . 8U 21+. R e la tiv e Enzymatic A c t iv i t ie s Per Unit P ro tein N itrogen During R..S. Developm ent................................................................................ 8H 25* Log P lo ts fo r D is tr ib u tio n o f N itrogen Per Plant During R .S . Developm ent......................................................... 8U 2 6 . The D is tr ib u tio n o f N itrogen Per Unit Weight o f Organism During R.S .Developm ent ............... 85 27. The D is tr ib u tio n o f N itrogen Per Plant During R.S. .................... Development 85 2 8 . The S o lu b le 87 AminoAcids of R.S. P lants During D evelopm ent.. x ii INTRODUCTION The problem of cause and e f f e c t r e la tio n s h ip s un derlying the d i f ­ f e r e n t ia t io n o f m orp h ologically complex str u c tu r e s has a ttr a c te d the a tt e n tio n o f man from th e tim e o f A r i s t o t le . U n til the proposal o f th e c e l l th e o r ie s o f S c h le id en and Schwann, and the work o f P asteur in th e mid 1800; s , however, l i t t l e r e a l progress was made toward under­ sta n d in g development . In f a c t , i t required the d is c o v e r ie s o f th ese men and oth ers to f i n a l l y la y to r e s t the sp e c u la tio n s o f the naturphilosophen concerning "preform ation," "spontaneous gen eration ," e t c . , which had s e r io u s ly hindered any em p irica l approach to the problem. Although tremendous s tr id e s have been made in embryology s in c e th a t tim e, th e in h eren tly -co m p lex , m u ltic e llu la r system s u t i l i z e d have impeded the development o f a com pletely s a t is f a c t o r y ex p la n a tio n o f the p ro cesses involved in growth. L it t l e enough i s known concerning the r e g u la tio n o f development in a s in g le c e l l . When a l l th e co m p le x ities and in te r r e la tio n s h ip s among the u n its in a m u ltic e llu la r str u c tu r e are added to g e th e r , th e d is ­ t in c t io n between th e p ro p erties due to the in d iv id u a l, and th o se due to th e p op ulation becomes most d i f f i c u l t . It would appear axiom atic th a t an understanding o f the p h y sio lo g ic a l and m orphological c a p a b il it i o f a s in g le c e l l should precede attem pts to e lu c id a te th e in te r a c tio n s among c e l l s . Such approaches have been, -and are being in c r e a s in g ly u t i l i z e d , p a r tic u la r ly the study o f is o la t e d plan t and animal c e l l s or t is s u e s in pure c u ltu r e . A second and somewhat sim pler experim ental 2 approach has in volved the study o f organisms which co n ta in one, or at most a few c e l l s at m atu rity. The a lg a e and fu n g i have shown them­ s e lv e s to be most s u ita b le fo r such s t u d ie s . A c a s e - in point i s th e eleg a n t work o f Hammerling (1953) concerning n u clea r-cy to p la sm ic r e la tio n s h ip s in th e m orphological development o f th e u n ic e llu la r , u n in u cle a te a lg a , A c eta b u la r ia . Many members o f the low er fu n g i a ls o appear to provide e x c e lle n t m a teria l fo r experim ental morphology. They can be grown under c o n tr o lle d experim ental co n d itio n s and d is p la y a d i s t i n c t but r e l a t i v e l y sim ple morphology. G enerally speaking, however, w ith a few important excep tion s the p o t e n t i a l i t i e s o f th e se organisms have not been e x p lo it e d . The g r e a te st percentage o f work in th e myco­ l o g i e s ! area has been confin ed to in v e s tig a tio n s o f th e environm ental c o n d itio n s n ecessa ry fo r growth and reproduction (Hawker, 1957) • I f a tt e n tio n i s turned to s tu d ie s o f morphogenesis at the c e ll u la r and p h y s io lo g ic a l l e v e l , very few examples can be found fo r the fu n g i. N otable among th e se is the worx o f N ickerson and h is c o lle a g u e s (1956, and r e fe r e n c e s th e r e in ) on th e biochem ical mechanisms o f c e l l d iv is io n in y e a s t s . Another important c o n tr ib u tio n has come from th e s tu d ie s o f Wright and Anderson (1959) on biochem ical d if f e r e n t ia t io n in th e slim e mold D ict.yostelium d isc o id iu m . Probably the most thorough approach, from the stan d p oin t o f a broadly b io lo g ic a l study c lo s e ly coordinated w ith a biochem ical in v e s tig a tio n o f d if f e r e n t ia t io n , has been th a t o f Cantino and co-workers (1951-1959) w ith th e aquatic Phycomycete, B la s t o c la d ie lla e m e r so n ii. In a d d itio n to the in t r i n s i c knowledge gained fo r the p a r tic u la r organism , each o f the s tu d ie s mentioned has 3 helped to provide in form ation n ecessa ry fo r an understanding o f develop^m ental phenomena common to the c e l l s o f many., or perhaps a l l , organism s. h LITERATURE REVIEW Morphogenetic S tu d ie s o f B la s t o c la d ie f la em ersonii The sim p le aqu atic fu ngus, B la s t o c la d ie lla em e rso n ii, was is o la t e d in pure c u ltu r e and i t s l i f e h is t o r y and development d escrib ed by Cantino (1951)* and Cantino and Hyatt (1953b). A fte r an i n i t i a l period o f m o t ilit y the sm a ll, u n if la g e l la t e spores o f t h is fungus s e t t le ' down, r e tr a c t t h e ir f l a g e l l a , and germ inate in a b ip o la r fa s h io n . The lower p o r tio n o f the growing p la n t develops in to a s i n g l e - c e l l e d , branching, r h iz o id a l system . The upper p o r tio n , a ls o c o n s is t in g o f a s in g le m u lti- n u c le a te c e l l , develops in to e ith e r a c o lo r l e s s , p a p illa t e , zoosporangium w ith .a t h in , c h itin o u s w a ll (r e fer r ed to as an ordinary c o lo r le s s or O.C. p la n t ) , o r , a brown, m elanin-pigm ented, th ic k -w a lle d , p itte d r e s is t a n t sporangium ( R. S. ) c o n ta in in g conspicuous l i p i d g lo b u le s . When th e mature p lan ts o f e ith e r type are placed under s u ita b le con­ d it io n s , th e y d isch arge m o tile spores (swarmers) and thus complete the l i f e c y c le w ith no con ven tion a l in ter v e n in g sex u a l s t a g e . In c o n tr a st to the c lo s e ly r e la te d genus, B la s t o c la d ia , which re­ quired a carbon d io x id e atmosphere o f alm ost 100$ to form R.S. (Emerson and C antino, 19 )48 ) , B ia s to c la d ie .lla spontaneously produced such str u c tu r e s under the crowded co n d itio n s in second g en era tio n c o l o n ie s . When th e c o n d itio n s n ecessa ry fo r R.S. form ation were stu d ie d , i t was found th a t e ith e r calcium carbonate or sodium bicarbonate induced the development o f R. S. It was fu r th e r e sta b lis h e d th a t on c a se in h y d ro ly sa te medium, where bicarbonate alone would not s u f f i c e , both 5 a -k e to g lu ta r a te and c it r a t e in creased the percentage o f R . S . formed, and th at a r s e n it e and sem i-carb azid e (both in h ib it o r s fo r the decarboxy­ la t i o n o f a -k e to g .lu ta r a te ) could rep la ce the organic acid requirem ent (C antino, .1951) • Having acquired the a b i l i t y to c o n tr o l the pathway o f developm ent, a n e a r ly id e a l system was a v a ila b le fo r stu d yin g th e m etab olic in t e r ­ a c tio n s involved in th e ontogeny o f the organism . The bicarbonate stim u lus w a s required fo r on ly t h r e e - f if t h s o f th e gen eration tim e o f th e R.S. p la n tsj co n v e rse ly , i t could n o t induce R. S, d if f e r e n t ia t io n in O.C. p la n ts a f t e r t h r e e - f if t h s o f t h e ir g en era tio n tim e had elap sed (C antino, 1 9 5 2 ). These r e s u l t s , to g e th er w ith th ose from p erm ea b ility s t u d ie s , le d to th e in te r p r e ta tio n th a t the e f f e c t o f th e bicarb on ate was to block d eca rb o x y la tio n r e a c tio n s in th e t r i ­ ca rb o x y lic acid c y c le , and th a t the r e s u lt in g p ile -u p o f in term ed ia tes caused a .s h i f t in the m etabolic p a ttern s toward R. S. developm ent, i . e . , th ic k e r w a ll, in creased pigment, l i p i d s y n th e s is , e t c . Et was fu rth e r su ggested th a t th ese changes le d to a gradually in c r e a sin g im p erm eability to both in te r n a l and e x te r n a l bicarbonate io n s . At approxim ately th r e e - f i f t h s of the gen eration tim e th e system was assumed to become autoc a t a ly t ic due to th e in creased r e te n tio n o f m eta b o .lica lly produced bicarbonatej removal o f th e e x te rn a l stim ulus a ft e r t h is sta g e could no lon ger r ev e r se th e p rocess (C antino, 1952). A com parative survey o f c i t r i c acid c y c le enzymes in th e w ild -ty p e O.C. p la n ts o f B la s t o c l a d ie ll a , and in an orange mutant (B.E.M.) derived therefrom , provided a d d itio n a l inform ation used to support the th eo ry 6 th at b icarb on ate helped to slow down and, w ith th e a s s is ta n c e o f oth er p u llin g r e a c tio n s , r ev e r se th e Krebs c y c le at the a -k e to g lu ta r a te sta g e (C antino, 1953; Cantino and Hyatt 1953b, 1953c). Most o f th e enzymes norm ally a s so c ia te d w ith th a t o x id a tiv e system were found to be presen t i in O.C. hom ogenates, in clu d in g : a c o n ita s e , a TPN s p e c if ic i s o c i t r i c dehydrogenase, a DPN or TPN coupled a -k e to g lu ta r a te o xid ase system , su c c in o x id a s e , fum arase, a DPN s p e c if ic m alic dehydrogenase, and cytochrome o x id a se . The. B.E.M. mutant, however, which co u ld 'n o t form R. S. p la n ts , even in the presence o f b ica rb o n a te, lacked both a c o n ita se and a -k e to g lu ta r a te oxid ase a c t i v i t y . The f a ilu r e o f B.E.M. to respond to b icarbonate was th e r e fo r e in ter p r e te d as b ein g due to th e absence o f th ose two enzymes in tim a te ly a s so c ia te d w ith the two s u c c e s s iv e decar­ b o x y la tio n s presumed to be th e s i t e s o f a c tio n . To stren g th en t h e ir th eo ry , C antino, jet aL., sought to con n ect, in a cause and e f f e c t manner, what had been d isco v ered concerning the bicarb on ate ’’t r ig g e r mechanism” w ith c e r ta in o f the m orphological mani­ f e s t a t io n s o f the R.S. p la n t. Mien t h a l l i were grown on bicarbonate medium c o n ta in in g th io u rea (an in h ib it o r o f the co p p er-co n ta in in g polyphenol o x id a s e s ), R. S. p la n ts were formed which were devoid of m elanin, but normal in a l l oth er r e s p e c ts (C antino, 1953)* This demon­ s tr a te d th a t a p rocess concom itant w ith development could be uncoupled from i t without oth erw ise d is r u p tin g i t s normal p r o g r ess. It was a lso e s ta b lis h e d in v i t ro th a t a wall-bound polyphenol o xid ase found in R.S. lR efer to Appendix t fo r the a b b rev ia tio n s used in t h is t h e s i s . 7 and presumably in volved in m elan ogen esis, could not be d e te c ted in O.C. p la n ts , thus in d ic a tin g i t s appearance, de novo, as a r e s u lt o f the bicarb on ate in d u c tio n (Cantino and H oren stein , 1955) • This th io u r e a - s e n s i t i v e , c y a n id e -in s e n s itiv e system not o n ly mediated e le c tr o n tr a n s­ port. between su b s tr a te ( e . g . , ty r o s in e and c a te c h o l) and oxygen, but a ls o between s u b str a te and TPN, but not DPN. A lp h a -k eto g lu ta ra te a lo n e , or th a t compound plu s b ica rb o n a te, always stim u la ted the r e a c tio n . I t was su ggested th at the e f f e c t was due to coupling between the re-;, d u c tiv e ca rb o x y la tio n o f c -k e to g lu ta r a te , y ie ld in g o x id ized TPN, and a "quinone oxidase" r e a c tio n in the polyphenol oxidase system ( i . e . , c a ta ly z in g the o x id a tio n o f o-quinone to hydroxy-o-quinone) reg en era tin g reduced TPN. The report th a t R. S. p la n ts contained an a c tiv e i s o c i t r i c dehydro­ g en ase, but a fe e b le s u c c in ic dehydrogenase and no a -k e to g lu ta r a te o xid ase or cytochrome o xid ase a c t i v i t y , demonstrated th a t th er e had been, as proposed, important s h if t s in th e c i t r i c acid c y c le enzymes o f th e R. S. (Cantino and H oren stein , 1955)- A bicarbonate-indu ced in ­ c r e a se in th e a -k e to g lu ta r a te content o f O.C. p la n ts was a ls o c o n s is te n t w ith the proposed a c tio n o f th a t io n , i . e . , in h ib it io n o f th e k e to -a c id d ecarb oxylation ('Cantino, 1 9 5 6 ). The presence o f r -c a r o te n e in the B.E.M. mutant (Cantino and H yatt, 1953b) and in the R. S. o f th e w ild type (Cantino and H o ren stein , 1956) provided an in t e r e s t in g p o s it iv e c o r r e la tio n between th e presence o f th e pigment and le s io n s in th e Krebs c y c le . s is of Presumably, however, th e synthe­ r -c a r o te n e could not have r e su lte d d ir e c t ly from the r e v e r sa l 8 o f th e a -k e to g lu ta r a te d ecarb o x y la tio n s in c e t h is enzyme was absent from B.E.M. The important d is c o v e r y -o f lig h t stim u la ted growth and l i g h t stim u la ted carbon d io x id e f ix a t io n in B ia s t o c la d ie lla helped to fu rth e r c l a r i f y the r o le o f bicarbonate in R.S. form ation (Cantino and Horen­ s t e i n , 1 9 ^ 6 ). Tracer s tu d ie s w ith uniform ly la b e le d g lu c o se -C 14 and b ica rb o n a te-C 14’were undertaken w ith preformed O.C. p la n ts incubated in th e lig h t and in th e dark. The most s ig n if ic a n t r e s u lt s were: (a) g lu c o se-C 14 gave r is e to la b e le d glutam ic and a s p a r tic a cid s lo n g b efore any d e te c ta b le r a d io a c t iv it y appeared in th e c i t r i c , acid c y c le compoundsj (b) Carbon-lit la b e le d bicarbonate was r a p id ly incorporated in th e Krebs c y c le compounds and, in p a r tic u la r , l i g h t caused a marked in c r ea se in la b e le d su c cin a te and a decrease in th e la b e le d a -k e to g lu ta r a te , as compared to dark c o n tr o ls , and (c ) a compound t e n t a t iv e ly id e n t if ie d as o x a la te acquired a s ig n if ic a n t q u a n tity o f C14. in v it r o experim ents w ith R.S. homogenates e x h ib ited a s lig h t lig h t in h ib it io n o f th e i s o c i t r i c dehydrogenase a c t i v i t y and, c o n v e r se ly , a s tim it a t io n by l i g h t o f i t s r e v e r sa l in the presence o f a -k e to g lu ta r a te , b ica rb o n a te, and reduced TPN. When th e l a t t e r was ca rried out u sin g b ica rb o n a te-C 14, th e fix e d Carbon-II4. was found in a -k e to g lu ta r a te , o x a la te , i s o c i t r a t e , and a s lig h t amount in s u c c in a te . When the same e x tr a c ts were incu­ bated w ith s u c c in a te , b ica rb o n a te-C 14, and reduced TPN, f i x a t io n of carbon-H 4 a ls o occu rred. I t was presumed, but not e s ta b lis h e d un- e q u iv o c a b lly , that the su c c in a te was carb oxylated to produce a -k e to ­ g lu t a r a t e . [n the l i g h t , reduced DPN y ie ld e d only o n e - f if t h the 9 f ix a t io n obtained w ith reduced TPN in t h is system , and in th e dark no f ix a t io n occurred w ith the former n u c le o tid e . Two im portant co n clu sio n s were drawn from the above o b s e r v a tio n s . F i r s t , the c i t r i c acid c y c le seemed an u n lik e ly path le a d in g to the s y n th e s is o f glutam ate and a s p a r ta te . Second, l i g h t in some way stim u­ la te d the r ed u ctiv e c a rb o x y la tio n o f a -k e to g lu ta r a te and th e subsequent clea v a g e o f the i s o c i t r a t e so formed to y ie ld su c c in a te and a C2 com­ pound such as g ly o x a la t e . The su c cin a te was a ls o presumed to undergo a l i g h t stim u la ted r ed u c tiv e ca rb o x y la tio n , v ia an undetermined TPN dependent system , to produce a -k e to g lu ta r a te and com plete the c y c le . This th eory has r e c e n tly been strengthened by"the i s o la t io n from B la s t o c la d ie lla (R. S. and O.C.) o f th e enzyme, i s o c i t r i t a s e , which c le a v e s i s o c i t r a t e to produce equimolar q u a n titie s o f su c cin a te and g ly o x a la te (McCurdy, 1959) . The dem onstration in v iv o th a t su c c in a te and g ly o x a la te could s u b s tit u te fo r th e e f f e c t o f l i g h t and bicarbonate served to fu rth e r support the in te r p r e ta tio n given above i,Cantino and H oren stein , 1999) • It was a lso e sta b lis h e d th a t the g ly o x a la te was r a p id ly converted to g ly c in e by transam ination w ith a la n in e , th ereby p rovid in g a p u llin g r e a c tio n fo r th e whole 3 . K . I . (.su c c in a te -k eto g lu t a r a t e - i s o c .itr a te ) c y c le (McCurdy, 1999) • A p o s s ib le co n n ectio n , v ia the use o f g ly c in e fo r thymine b io s y n t h e s is , has r e c e n tly been proposed between th e S . K . I . c y c le and l i g h t stim u la tio n o f n u c le ic acid s y n th e s is and nucle-ar reproduction in B la s t o c la d ie lla (Turian and C antino, I 9 6 0 ). The s ig n ific a n c e o f the preceding o b serv a tio n s w ith r esp e c t to th e form ation o f R.S. was as fo llo w s : (1) i t helped to v e r if y th e scheme 10 proposed fo r B .S . in d u c tio n by p rovid in g a pathway which could operate in the absence o f most o f the c i t r i c acid c y c le enzymes; (2) i t in d ic a te d a d d itio n a l p o in ts where in term ed ia tes could accumulate due to th e b i­ carbonate tr ig g e r e f f e c t and, in so doin g, in flu e n c e oth er r e a c tio n sequences; and (3 ) i t s tr o n g ly suggested th a t l ig h t stim u la ted growth and carbon d io x id e f i x a t i o n , and R. S. in d u c tio n , were a c tu a lly two d if fe r e n t e x p ressio n s o f th e p o t e n t ia lit y o f a s in g le enzyme system in respon se to d if fe r e n t environm ental co n d itio n s . The a b i l i t y o f l i g h t to reduce the co n cen tra tio n o f bicarbonate n ecessa ry to induce the form ation o f R. S. was taken as a confirm ation o f th is in te r p r e ta tio n (C antino, .1957) • The id e n t if ic a t io n o f c h it i n as the primary c e ll - w a ll c o n s titu e n t in B la s t o c la d ie lla was e sta b lis h e d by glucosamine and a c e ta te an alyses fo llo w in g a cid h y d r o ly sis o f p u r ifie d w a ll m a teria l (C antino, L o v ett, and H o ren stein , .1957)* Homogenates o f R.S. and O.C. p la n ts contained comparable g lu c o sa m in e -a c e ty la tin g a c t iv it y in system s c o n ta in in g a c e t a te , coenzyme-A, and ATP (or acety lm eth io n in e and ATP). In a l l ca ses the wall-bound a c t iv it y - w it h the non-phosphorylated glucosamine was low . In c o n tr a st to a c e t y la t io n , th e c h itin a s e a c t i v i t y o f O.C.- supernatants w ith f in e l y d iv id e d , p u r if ie d , B la s t o c la d ie lla c h it i n proved to be 3 to 7 tim es th a t o f the P . S . In a d d itio n to th e change in c h it in n itr o g e n , a n a ly ses o f the t o t a l acid so lu b le and water so lu b le n itro g e n fr a c tio n s o f both R. S. and O.C. p la n ts had in d ic a te d a con sid era b le in t r a c e llu la r r e d is t r ib u tio n o f the n itrogen ou s c o n s titu e n ts between the two plant forms (C antino, L ° v e tt, 11 and H o ren stein , 1957) . With th e ex cep tio n o f th e se a n a ly s e s, and a prelim in ary report concerning d iffe r e n c e s in the e le c t r o p h o r e t ic a lly se p a r a b le , s o lu b le p ro tein s in the two plant ty p e s , l i t t l e work had been done on th e n itro g e n metabolism o f B la s t o c l a d i e l l a . However, i t appeared almost c e r ta in th a t the changes which occurred during d i f ­ f e r e n t ia t io n in v o lv ed fundamental tran sform ation s in n itro g e n m etabolism . The research reported h e r e in was in i t ia t e d to determ ine, in so fa r as p o s s ib le , i f t h is were s o . I t seemed l i k e l y th a t an understanding of such changes would h e lp c o n sid era b ly in the u ltim a te form u lation o f a coherent exp la n a tio n o f growth and d if f e r e n t ia t io n in B la s t o c l a d i e l l a a t th e c e ll u la r and organism al l e v e l s . Glucosamine S yn th etase and the B io sy n th e sis o f C h itin Although the presence o f c h i t i n in the c e l l w a lls o f fu n g i, and th e f a c t th a t i t contained n itr o g e n , was e sta b lis h e d as e a r ly as 1811 by Braconnot, and by L assaigne in 18H3 (T racey, .1955) r e s p e c t iv e ly , i t s b io s y n th e s is r eceiv ed l i t t l e a tte n tio n u n t i l recen t years . Most o f the work reported during th e in te r v e n in g period was concerned w ith its ' i d e n t if ic a t io n and i s o la t io n from variou s s o u r c e s. Two fa c to r s are perhaps most r e sp o n sib le fo r th e contemporary in t e r e s t in the s y n th e tic r e a c tio n s o f hexosamine compounds . The f i r s t i s the r e l a t i v e l y recen t a v a i l a b i l i t y o f the biochem ical techniques fo r stud ying enzym atic r e ­ a c tio n s w ith m ic r o q u a n tities o f biochem ical compounds. The second i s th e u b iq u itou s presence o f hexosamines in th e m ucopolysaccharides which are b ein g in t e n s iv e ly stu d ied in con n ection w ith the b io ch em istry o f b lood , and a r t h r it ic d is o r d e r s . 12 Whatever th e subsequent f a t e o f th e hexosam ines, most o f the b io ­ s y n th e tic sequences u t i l i z i n g th e se compounds appear to req u ire thes y n th e s is o f glucosam ine phosphates as a f i r s t s t e p . Harpur and Q uastel (19U9) demonstrated the p h osp horylation o f d-glucosam ine (GA) by b ra in e x tr a c ts in th e presence o f ATP. Although the products were n ot i s o ­ la t e d , the M ich aelis c o n s ta n ts , and th e co m p etitio n observed between d-glu cosam in e, d -g lu c o s e , and d -fr u c to se in m ixtures su ggested th a t a l l th re e compounds were phosphorylated by the same enxyme. N -a c e ty l- glucosam ine (AG) was u n a ffe c ted by th e enzyme and acted as a co m p etitiv e in h ib it o r fo r a l l th ree compounds. A s im ila r s y n th e s is was reported w ith B ak ers-yeast preparations by Grant and Long ( 19 52 ) . These authors a ls o found evidence fo r com p etition between GA and g lu co se fo r the same enzyme. The a b i l i t y o f hexokinase to phosphorylate GA was esta b ­ lis h e d by Brown (1951) u t i l i z i n g a c r y s t a llin e y e a st enzyme. Analyses o f the r e a c tio n p r o d u c t(iso la te d by barium f r a c t io n a t io n ) , in d ic a te d th a t i t was a monophosphate p o sse ssin g reducing p ro p e r tie s and a fr e e amino group. The p o s itio n of the phosphate e s t e r at carbon-6 was a sce r ta in e d by comparative sodium m etaperiodate o x id a tio n s w ith GA and d -g lu c o s e -6 -phosphate (G- 6-P). The str u c tu r e was e sta b lis h e d to be th a t o f d- glue os amine-6- phosphate (GA-6-P). L e lo ir and C ardini (1953) reported th a t p a r t ia lly p ir i f ie d prepara­ tio n s from Neurospora c ra ssa ca ta ly zed th e sy n th esis o f GA-6-P from f r u c t o s e - 6 - phosphate (F -6 -P ), or G-6-P, and glutam ine. The enzyme, which had been p u r ifie d c a . 8 - f o ld by aoetone fr a c tio n a t io n , had a s p e c i f i c a c t i v i t y o f 0-3 pM GA/mg. p r o te in /h r ., and tem perature and pH optima o f 30° C. and 6 .I4 to 6 . 8 , r e s p e c t iv e ly . Both the hexose phos­ phates and glutam ine e x h ib ite d a s t a b i l i z i n g e f f e c t on th e rath er l a b i l e enzyme. No c o -fa c to r requirement could be demonstrated f o r the r e a c tio n , nor could any o f a number o f s tr u c tu r a lly r e la te d compounds s u b s t it u t e fo r e it h e r of th e h e x o se s, or rep la ce glutam ine as th e amino donor. Blum enthal, e t a l . (1 9 5 5 '), w ith more h ig h ly p u r ifie d prepara­ tio n s demonstrated th a t F - 6 -P was the primary su b str a te fo r th e Neurospora enzyme ( glu tam in e-F - 6 - P transam idase, c . f . Comb and Roseman, 1958) • Crude e x tr a c ts from a P e n ic illiu m s p e c ie s had the same r eq u ir e ­ ments . P o g e ll and Gryder (1956, 1957a,b) p a r t ia l ly p u r ifie d an enzyme (am in otran sferase) from r a t l i v e r wnich u t i l i z e d G-6 -P and glutam ine fo r GA-6-P s y n th e s is . The approxim ately 2 to 3 - f o ld p u r if ic a tio n a tta in e d did not e lim in a te enzyme a c t i v i t y w ith F - 6-P as s u b s tr a te , but m erely reduced i t as compared to th a t w ith G-6-P . The two hexose phosphates were eq u a lly a c tiv e in th e crude hom ogenates. The system had a pH optimum o f 7 .I4 and, as w ith the Neurospora r e a c tio n , no co­ f a c to r requirement could be found. This enzyme a ls o resembled the Neurospora one in th a t th e very l a b i l e enzyme could be s t a b iliz e d by the a d d itio n o f hexose ph osp hates. Poge.ll and Gryder considered the greater s t a b i l i t y obtained w ith G-6 -P , as compared w ith F - 6 -P , an in d ic a tio n o f i t s r o le as a more immediate precursor fo r th e r e a c tio n . N either th e Neurospora, nor th e r a t l i v e r enzyme was su b jected to more than a p a r tia l p u r if ic a tio n and, th e r e fo r e , no c r i t i c a l s tu d ie s have been made to e lu c id a te e ith e r th e mechanism or the eq u ilib riu m of th e •Ill r e a c tio n . The p a u c ity o f d e f i n i t i v e inform ation concerning th e se GA-6 -P s y n th e s iz in g system s may w e ll be a r e f le c t i o n of th e i n s t a b i l i t y o f the enzyme p ro tein s and. th e consequent d i f f i c u l t i e s in h eren t in t h e ir p u r if ic a t io n . The same problem arose in the p u r if ic a tio n o f the B la s t o c la d ie lla enzyme. The conversion of GA to F - 6-P and ammonia has been stu d ied exten ­ s i v e l y in E sch erich ia c o l i (Soodak, 1955; Faulkner and Q u a stel, 1956; W olfe, _et _al. , ,1956a,b ,c, 1957, 1959; Roseman, 1956; Comb and Roseman, 1956, 1958) , in Aerobactor clo a ca e (Imanaga, e t a l . , 1 9 5 7 a ,b ), and in brain e x tr a c ts (Faulkner and Q u a stel, 1 9 5 6 ). I t was e sta b lis h e d th a t a common pathway in th e th ree organisms occurred through th e phos­ p h o ry la tio n of GA w ith ATP to produce GA-6-P , and subsequent deam ination o f th a t compound to y ie ld F- 6 -P and ammonia. The u b iqu itous d is t r ib u t io n o f h exok in ase, and i t s demonstrated a b i l i t y to .p h o sp h o ry la te GA make i t le g itim a te to expect th e presence o f the aforem entioned system in many organism s. L e lo ir and Cardini (1956) reported that preparations from hog kidney ca ta ly zed GA-6 -P form ation from F - 6 -P and ammonia. The r e v e r s ib le r e a c tio n required N -a cety lg lu co sa m in e- 6 -phosphate (AG-6-P ) as a c o -fa c to r and d isp la y ed optim al a c t i v i t y a t pH 8 .I4 . An eq u ilib riu m con stan t o f c a . 0 .1 2 to 0 .18 was given fo r the r e a c tio n 1F - 6-P + NH3 GA-6- P ) , i . e . , i t had a stro n g tendency toward the production o f F- 6 -P and ammonia. Comb and Roseman (1956) showed th a t the E. c o l i deaminase was a ls o r e v e r s ib le in p a r t ia l ly p u r ifie d p r e p a r a tio n s, The same 15 workers ( 1958 ) made a com parative study w ith p u r ifie d deaminases from hog kidney and E. c o l i . The r e s u lt s in d ic a te d th a t th e r e a c tio n mechanism was th e same fo r both enzymes and that th e AG-6 -P stim u la te d , but was not req uired f o r , th e r e a c tio n . The d ir e c t p a r tic ip a tio n of AG-6 -P in the co n v ersio n was ruled out by the use o f i s o t o p ic a lly la b e le d compounds. Although F - 6 -P and ammonia form ation was g r e a tly favored, the r e v e r s i b i l i t y was con­ firm ed f o r both deam inases. I t was, however, demonstrated th a t a rapid s y n th e s is o f AG-6-P from F - 6-P'and ammonia could be obtained w ith both enzymes i f the r e a c tio n was coupled w ith a p u r ifie d GA-6 -P a c e ty la s e (D avidson, Blum enthal, and Roseman, 1957) and acetyl-coen zym e-A . In summary, GA-6 -P has been shown to be sy n th esized by at l e a s t fou r d if fe r e n t enzyme system s: (1) from GA and ATP in a k in a se r ea c tio n ; ( 2 ) by the glu tam in e-F - 6- P transam idase system; ( 3 ) by the aminotrans­ fe r a s e r e a c tio n w ith G-6 -P and glutam ine; and (Ij.) by a r e v e r sa l o f the deaminase system s . A second name, glucosamine sy n th e ta s e , su ggested by Roseman (p erson al communication) fo r r e a c tio n (2) w i l l be used in t h is paper fo r th e sake o f convenience. Although th e known b io s y n th e tic pathways th a t ram ify from GA-6 -P are ra p id ly e n la r g in g , only th o se p erta in in g to c h it in s y n th e s is w i l l be summarized h e r e . The a c e t y la tio n of GA-6 -P w ith acetyl-coenzym e-A to form AG-6 -P has been demonstrated in B akers-yeast (Brown, 1 9 5 5 ), Neurospora ( L e lo ir and C ardini, 1953; D avidson, Blum enthal, and Roseman, 1 9 5 6 ,1 9 5 ? ), Pe .n ic illiu m , S trep tococcu s „ rabbit m uscle, and human l i v e r (D avidson, Blum enthal, and Roseman, 1 9 5 7 ). The AG-6 -P has been shown 16 to undergo a phosphoacetylglucosam ine mutase r e a c tio n (AG-6 -P AG-l-P) in Neurospora (L e lo ir and Card'ini, 19935 R e is s ig , 1 9 9 6 ), and in hog kidney (L e lo ir and C ard in i, 1 9 9 6 ). E ith er g lu c o s e - 1 , 6 -d ip h osp h ate (G -1,6-DP) or N -a c ety lg lu c o sa m in e -1 , 6 -diphosphate could serv e as the c o -fa c to r fo r th e co n v ersio n . R e is s ig p a r t ia l ly separated the enzyme from th e phosphoglucomutase p resen t in Neurospora e x tr a c ts and found a r a tio o f 86 % AG-6 -P : ll 4.% AG-l-P a t e q u ilib riu m . He a ls o observed th e form ation o f AG-1,6 - DP when the enzyme was incubated w ith AG-l-P and G -1,6-DP. Maley and Lardy (1996) e sta b lis h e d th e a b i l i t y o f r a t l i v e r p reparations to form u rid in ed ip h osp h ate-N -acetylglu cosq m in e (GDPAG) from AG-l-P and (JTP. UDPAG was f i r s t is o la te d from B ak ers-yeast by Cabib, L e lo ir , and C ard in i, 1993) and has a lso been found in r a t l i v e r (Smith and M ills , 1993) and mung bean (Solms and H assid , 1997) • Of p a r tic u la r in t e r e s t was th e dem onstration by G laser and Brown (199?a,b ) that> a p a r tic u la te fr a c tio n from Neurospora could in corp orate th e AG m oiety o f GDPAG in to c h i t i n - l i k e compounds. The r e a c tio n was a c c e le r a te d by the presence o f high m olecular weight c h ito d e x t r in s . Although th e r e a c tio n s d escrib ed in th e preceding paragraphs have been stu d ied in a v a r ie ty o f organisms and t is s u e s a l l but one have been found in the m ycelia o f N eurospora. Perhaps i t can be assumed, th e r e fo r e , th at the whole b io s y n th e tic sequence le a d in g to c h i t i n , as o u tlin e d , occurs in Neurospora and, by analogy, in oth er fu n gi producing c h itin o u s p o ly sa c c h a r id e s. Some o f the hexosamine compounds and th e ir in te r a c tio n s are in d i­ cated sch em a tic a lly in Figure 1 fo r th e convenience o f the rea d er. 17 F igure 1 Sugar-Phosphate and Hexos amine-Phosphate In terco n v ersio n s Glucose + ATP ATP +• Fructose F-6-P 0-6-P N. c ra ssa Rat l i v e r + glutam ine GA + Pi • N. c r a ssa Hog kidney E. c o l i NH Yeast + G-1,6-DP N. c ra ssa P e n ic illiu m Strep tococcu s Rabbit muscle Human l i v e r 4 f UTP AG-6-P AG Rat liv e r Ac-Co A Yeast Brain E. c o l i F-6-P + NHo + A ceta teTT^— ^ Hog kidney N. cra ssa 'f AG-l-P + UTP (UDP-acetylmannosamine) f UEP + Acetylmannos amine GA-l-P Rat L iver n u c le i V UDPGA + G-1 , 6 , -DP AG-1,6 -DP N. c ra ssa A Rat l iv e r r UDPAG ^ B . s u b tilis U D P-acetylgalactosam ine N. c ra ssa r C h itin UDP 4 a c e t y lg a la c t o s ­ amine 18 MATERIALS AND METHODS C ulturing and H arvestin g Procedures To o b ta in rep rod u cib le experim ental r e s u lt s i t was n e c essa ry to sta n d a r d iz e , in so fa r as p o s s ib le , th e c u ltu r in g and h a r v e stin g tech n iq u es used in th is stu d y . For t h is reason the procedures u t i l i z e d have been d e scrib ed in co n sid era b le d e t a i l . Stock C ultures .— A ll c u ltu r e s o f B la s t o c la d ie lla were grown on a b a s ic medium ;PYG) co n ta in in g : 1 .2 5 gm. D ifco y e a st e x tr a c t, 1 .2 5 gm. D ifc o peptone, and 3*0 gm. g lu co se per l i t e r o f water (Cantino and H o ren stein , 1955)*. To t h is b a sic medium e ith e r B rom -cresol purple (BCP), sodium bicarb on ate plus BCP, or 2% D ifc o agar was added as d escrib ed below . Stock c u ltu r e s were m aintained as c o lo n ie s o f R .S . p la n ts on P e tr i p la te s o f PYG a gar. Swarmer in o cu la were obtained by p la c in g b lock s o f agar bearing R .S. in a sm all volume o f s t e r i l e water ^Barner and C antino, 1 9 5 2 ). A fter swarmer d isch a rg e '(5 To 15 hours depending upon th e h is t o r y o f th e c u lt u r e ) , p la te s or f la s k s were in o cu la ted w ith the su sp en sion s u sin g standard b a c t e r io lo g ic a l tech n iq u es . C ultures fo r Zoospore H arvests and Large S c a le .In o c u la .— Ten or 15 cm. diam eter P e tr i d ish e s of PYG agar were in o cu la ted w ith heavy swarmer su sp en sion s and incubated at 20 or 2l|.0C . fo r 20 or 16 hou rs, r e s p e c tiv e ly j th e se c o n d itio n s produced mature f i r s t gen eration p la n ts ready to d is ­ charge. The p la te s were then flo o d ed w ith approxim ately 10 m l. o f 19 s t e r i l e w ater and allow ed to stand at room tem perature fo r 15 to 60 minutes fo r e x te n s iv e spore d isch a rg e to occur- The r e s u lt in g suspen­ sio n s were then used to in o c u la te liq u id c u ltu r e s > vse e below ) and sto c k p la t e s , or th ey were h arvested according to the method o f McCurdy (,1959) fo r a n a ly t ic a l s t u d i e s . S ta n d a r d iz a tio n o f Zoospore In o c u la .— A standard curve fo r the . c o n c en tr a tio n o f swarmer su sp en sion s was e s ta b lis h e d by determ ining the ab sorp tion o f a d ilu t io n s e r ie s in a Klett-Summerson p h o to e le c tr ic c o lo rim eter at 5.20 mp, corrected fo r the a b sorp tion o f the suspending medium, .and by t o t a l v ia b le counts on th e s e same su sp en sion s u sin g PYG _5 agar. The slo p e o f the curve obtained was 2 .1 5 x 10 , or th e number c o f swarmers per m i l l i l i t e r = co rrected a b s o r p tio n /2 .15 x 10 . The numbers o f v ia b le swarmers used f o r in o c u la tin g f la s k s were r o u tin e ly determ ined as d e s c r ib e d ■above. L iquid C u ltu r e s .--F o r preparation and p u r if ic a tio n o f enzymes, c u ltu r e s o f 0 .C . p la n ts were grown in e ith e r 3~ or 5 - l i t e r Erlenraeyer f la s k s c o n ta in in g 1 .5 or 3 *5 l i t e r s , r e s p e c t iv e ly , o f PYG medium and 10 5 % BCP. These fla s k s were equipped w ith an a e r a tio n tu b e, an inocu­ l a t i o n tu b e, and a side-arm t e s t - t u b e co n ta in in g 1 N potassium hydroxide fo r n e u tr a liz in g c u ltu r e s during growth (Cantino and H o ren stein , 1955)I n i t i a l l y th e 0 .C . p la n ts were grown w ith a era tio n fo r 3 to 5 days at, room tem perature, i . e . , fo r se v e r a l g e n e ra tio n s. At the tim e o f h a rv est 5 m l. o f th e flo c c u le n t su sp en sion o f p la n ts in each c u ltu r e was used to in o c u la te a new f l a s k . For the major p o rtio n o f the enzyme 20 p u r if ic a t io n work, how ever, m ature, h e a lth y , f i r s t g en era tio n p la n ts were obtained by in o c u la tin g 1 . 5 l i t e r fla s k s w ith dense swarmer su s­ pensions and a e r a tin g the c u ltu r e s v ig o r o u s ly over l ig h t s (c a . 300 F.C .) a t room tem perature (2[|°C .) fo r 13 to 15 h o u rs. By t h is method n e a r ly synchronous, f i r s t g en era tio n c u ltu r e s were obtained (McCurdy, 1959) s in c e th e t o t a l p op u lation was placed in th e f la s k as swarmers, i . e . , a l l at th e same s t a g e .o f developm ent. For producing R .S . p la n ts f la s k s were s e t up in e s s e n t i a ll y the same manner as fo r 0 .C. c u ltu r e s excep t th a t th e a lk a l i tube was re­ moved, and sodium b icarbonate o f d if f e r e n t co n cen tra tio n s was added to th e PYG b r o th . However, la r g e s c a le synchronized c u ltu r e s fo r stu d yin g R.S* ontogeny were grown in 1 2 - l i t e r , fla t-b o tto m f la s k s c o n ta in in g .10 3 l i t e r s o f PYG b ro th , in d ic a to r , and 8 .9 x 10 M sodium bicarbonate b efo re a n to c la v in g . These f la s k s were a ls o equipped w ith a g la s s siphon tube connected by Tygon tu bing to a 's u c tio n apparatus fo r a s e p tic a l l y removing samples o f the c u ltu re at variou s tim e i n t e r v a l s . A ll R .S . c u ltu r e s excep t th ose intended fo r a 12. hour h a r v e st, were in o c u la te d w ith c a . 1*37 x 1 0 6 swarmers per l i t e r o f medium. This was th e optim al p op u lation d e n s ity fo r normal growth and adequate y ie ld fo r th e experim ental procedures em ployed. Twelve hour c u ltu r e s were in o cu la ted w ith ), were used as s o lv e n t s , th e l a t t e r fo r th e se p a ra tio n o f phosphate e s te r s in p a r tic u la r (L e lo ir and P a l l i d i n i , 1952) . The one-dim ensional Rf v a lu es fo r s e v e r a l compounds w ith d i f ­ fe r e n t s o lv e n ts and papers are tab u lated in Appendix I I I . Whatman # 1 paper was washed as fo llo w s: Large s h e e t s , serra ted at th e low er edge, were placed in p a irs in a la r g e chromatography c a b in e t. One hundred and f i f t y m l. o f 2 N a c e t ic a c id , c o n ta in in g 0 .0 2 $ sodium EDTA, was added to each trough and allow ed to m igrate u n t i l a l l o f i t had run o f f the s h e e t s . The paper was th en washed tw ic e w ith g l a s s - d i s t i l l e d water in th e same manner, and f i n a l l y d ried in th e h ood . Propanol, b u ta n o l, and propion ic acid were a l l d i s t i l l e d b efo re u se. The phenol used fo r q u a n tita tiv e chromatography was a ls o f r e s h ly d i s t i l l e d , w h ile th a t used fo r q u a lit a t iv e chromatography was a Merck reagen t grade o f c r y s t a ls , sto r ed in th e cold u n t il u sed . When i t was n e c essa ry to r e t a in the proper c o lo r i n t e n s i t i e s o f amino a cid sp o ts on chromatograms fo r la t e r o b serv a tio n or photography. 2h th ey were sprayed w ith 0 N n ic k e l s u l f a t e , which convert ed th e purple to a red c o lo r w ithout lo s s of' the c o r r e c t, r e la t iv e i n t e n s i t i e s o f shading (Khaba and El* k in , 195b) ■ the c o lo r s thus obtained were s t a b le fo r s e v e r a l months, i f p rotected from b right l i g h t . Q u a n tita tiv e Determinat io n o f Amino Acids by Chromatography. — In order to o b ta in q u a n tita tiv e e stim a tio n s o f amino a cid s in plant e x t r a c t s , a liq u o ts were chromatographed in th e tw o-dim ensional system d e sc r ib e d . F ollow ing chromatography, th e in d iv id u a l amino a cid sp o ts were cut out and determ ined sp e ctro p h o to m e tr ic a lly by th e ninhydrin method o f Landua and Awapara 1.-19U9. Awapara, .1959; s e e C antino, L o v e tt, and H o ren stein , 1957)* Curves prepared by tr e a tin g known q u a n titie s o f pure amino a cid s in th e same manner were used as referen ce sta n d a rd s. For q u a n tita tiv e determ in ation o f amino a cid s w ith one-dim ensional paper chromatography o f sim ple m ixtures, the method of Kay, e t a l . yl956) -was fo llo w e d w ith slig h t' m o d ific a tio n . Propanol-ammonia was s u b s titu te d fo r th e so lv en t used by the a u th ors, and sodium hydroxide was d e le te d from th e 0.5% ninhydrin spray fo r c o lo r development . Propanol-ammonia y ie ld e d e x c e lle n t sep a ra tio n o f the amino a cid s in enzyme r e a c tio n m ixtures w ith un iform ly low blank paper v a lu es . The sodium' hydroxide was recommended by Kay, e t a l . because i t enhanced th e c o lo r form ation w ith ta u r in e , but because i t was found to depress the c o lo r in t e n s it y w ith glucosam ine i t s use was u n d esira b le in th ese experim ents . For chromatography of enzyme r e a c tio n m ix tu res, t r ic h lo r o a c e t ic acid (TCA) was used as th e deprote i n i zing agent* at a f i n a l co n cen tra tio n of 5$. The TCA was removed b efo re chromatography by e x tr a c tio n w ith e th y l eth er in a sm all a l.l- g la s s continuous e x tr a c to r . E le c t r o ly t ic d e s a lt in g w ith a R.eco e l e c t r i c d e s a lte r improved the q u a lity o f the chromatography but caused se r io u s decom position o f glueam ine. Ex tr a c tio n o f S o lu b le Amino A c id s .—Wet mats o f plant m a teria l were placed in a vacuum d e s ic c a to r over calcium c h lo r id e im m ediately a f t e r h a r v e s tin g , and sto r ed at 0 to 2°C . u n t il thoroughly d ry . The dried m a teria l ^100 mg.) was ground to a f in e p a ste w ith a m ixture o f powderedg la s s , carborundum, and 80 $ eth a n o l { 2 ^ 0 mg. grinding m ixture eth a n o l per 100 mg. p la n t s ) . 3 m l. The m a teria l was ex tra cted th ree tim es (T o t. V o l. l l | m l.) and th e pooled e x tr a c ts taken to d ry n ess, e ith e r by removing most o f the s o lv e n t on a steam bath and then drying in vacuo over calcium c h lo r id e , or e n t ir e ly in vacuo w ithout h e a t. the f i n a l drying was ca rried out a t 0 to 1;°C. In both ca ses The ex tr a cte d amino acid s were f i n a l l y r e -d is s o lv e d in 2 .0 ml. o f e ith e r 20 $ eth a n o l or d i s t i l l e d w ater, th e e x tr a c ts cen trifu g ed i f n ecessa ry to remove in ­ s o lu b le r e s id u e , and a liq u o ts chromatographed tw o -d im en sio n a lly . P rotein D eterm in a tio n . —P ro tein s were estim ated by th e tu rb id om etric method o f Stadtman, e t a l . (1951)* w ith s lig h t m o d ific a tio n . The pro­ t e i n sample was d ilu te d to 3 ml. w ith 0 .5 M potassium c h lo r id e , 3-0 ml. o f 5$ TCA were added w ith m ixing, and the tu r b id ity measured a f t e r one minute in a K le tt—Summerson co lo rim eter w ith a 580 mp f i l t e r . p lo t was obtained in th e range 0 .1 to 1 .5 mg, p r o te in . A lin e a r C r y s ta llin e serum bovine albumin (Sigma Chem. C o.) was used as a p r o te in standard. 26 Determinat ion o f Ammonium Ion and T otal N itr o g e n . — Free ammonium io n was determ ined by d ir e c t N e s s le r iz a tio n w ith a commercial prepara­ t io n o f th e F o lin and Wu (1919). N e ssle r reagent (H arleco T’Dry-Pack” ) . The samples were N e s s le r iz e d , allow ed to stand fo r 20 m inutes, and measured in th e K lett—Summerson co lo rim eter w ith a U20 mp f i l t e r . T otal n itr o g e n was estim ated by wet d ig e s tio n o f organic m a ter ia ls w ith s u lf u r ic acid and hydrogen peroxide over a m icro-burner, u n t i l a c le a r s o lu tio n was obtained (Um breit, B u r r is, and S t a u ffe r , 1957)* The d i­ gested samples were then d ilu te d , the ex cess s u lf u r ic acid n e u tr a liz e d w ith sodium hyd roxid e, and the samples N e ssle r iz e d and measured in the same manner as fo r fr e e ammonium io n . Inorganic and T o ta l Phosphorus .— Inorganic phosphorus was d e te r ­ mined by th e method o f F isk e and Subbarow (,1925) • T otal phosphorus was estim ated by a m odified method u sin g p e r ch lo ric acid (and n i t r i c acid or hydrogen peroxide i f n e c essa ry ) to d ig e s t samples o f organic m a teria l A lle n , 19U0) • B efore c o lo r development w ith the F iske and Subbarow r e a g e n ts, th e e x ce ss p e r c h lo r ic acid was n e u tr a liz e d w ith sodium h yd roxid e. The com pleteness o f th e d ig e s tio n procedure was r o u tin e ly checked by the u se o f 3 -p h osp h o g ly ceric acid as a stand ard . E stim ation o f H exese-phosphates .— F ru cto se-6 - phosphate or m ixtures o f F-6-P and G-6-P were estim ated by the anthrone method o f Mokrasch ( 195U)• The commercial preparation o f g lu c o s e -6 -phosphate denydrogenase '(Sigma Chem. Co.) used to determ ine G-6-P in the presence o f F-6-P 2 ' i^Horecker and Wood, 1 9 5 7 ), contained phosphohexoisomerase a c t i v i t y . However, th e d if fe r e n c e in th e i n i t i a l r a te s was used to e s t a b lis h the presence o f th e isom erase in homogenat.es co n ta in in g F-6-P or G -6-P. Glucosamine D eterm in a tio n .— Glucosamine and GA-6 -P were o r ig in a lly determ ined by the Immers and Vasseur (19£2) m o d ific a tio n o f th e E lson Morgan r e a c tio n . However, t h is method did not produce e q u iv a len t c o lo r v a lu e s fo r GA-6 -P as compared to GA and w as, in a d d itio n , s u s c e p tib le to in te r fe r e n c e by m ixtures o f amino a cid s and sugars (H orow itz, tkawa, and F lin g , 1 9 5 0 }. The D isch e and Borenfreund ^1950) method f o r GA, in v o lv in g deam ination w ith n itro u s acid and co lo r form ation w ith an in d o le -h y d r o c h lo r ic acid m ixture, y ie ld e d eq u iv a len t c o lo r form ation w ith both hexosamines • The sugars and amino a cid s .in the r e a c tio n mix­ tu r e s an alyzed , produced on ly a n e g lig ib le amount o f c o lo r even when p resen t in la r g e e x c e s s , and t h is error was elim in a ted by running nondeaminated c o n tr o ls (hexosam ines do not produce c o lo r without p rio r d eam in ation ), and by determ ining the o p t ic a l d e n s ity a t two d if f e r e n t wave le n g t h s . For th is reason i t was p o s s ib le to perform th e D isch e r e a c tio n d i r e c t l y on r e a c tio n m ix tu res, and i t was used r o u tin e ly fo r a l l th e la t e r d e te r m in a tio n s. In order to determ ine th e amounts qf GA-6 -P and GA in m ixtures c o n ta in in g b oth , a micro method was d evised u t i l i z i n g paper chroma­ tography to s e p a r a te 1th e two compounds and a c o lo r im e tr ic d eterm in ation w ith th e D isch e r e a c tio n . M ixtures were chromatographed o n e-d im en sio n a lly on washed Whatman # 1 paper w ith propanol-ammonium hydroxide-wat e r . 'J'he lo c a t io n o f the hexos amine .sp o ts was e sta b lis h e d by spraying p a r a l le l. 25 d u p lic a te s t r ip s w ith n in h yd rin . The same areas were removed from the experim ental s t r ip s (in c lu d in g paper b la n k s), cut in to sm all p ie c e s , and th en placed .in 12 m l. c e n tr ifu g e tu b e s. The D ische r e a c tio n fu sin g o n e -h a lf the normal q u an tity o f rea g en ts) was then ca rr ie d out in th ese tu b e s, th e paper packed in th e bottom o f each tu b e, and f i n a l l y th e s o lu tio n s poured in to 5 m l. c e n tr ifu g e tubes ( t i g h t l y capped w ith parafilm to prevent evaporation) and cen tr ifu g e d fo r 3 minutes at 1600 x g . to remove the f i l t e r paper f i b e r s . The colored s o lu tio n obtained ( c a . 2 .^ m l.) was th en read in the Beckman Model DH spectrophotom eter w ith appropriate sta n d a rd s. Linear standard curves were obtained fo r both hexosamines in the range from 0 .0 1 to 0 . 0 5 jiM. C h itin A n a ly se s.— The c h it in content o f m a ter ia l d ried to constant w eight at 95° C. was analyzed by a m o d ific a tio n o f the methods of Hackman (19510 and Tracey (.1955) • Ike samples were powdered in a mortar and approxim ately 20 mg. tra n sferred to a 10 x 120 mm. Pyrex tu be. One m l. of 1 N sodium hydroxide was added to each tu b e, a sm all g la ss bulb-cond en ser placed in th e mouth to prevent e x c e s s iv e ev a p o ra tio n , and th e con ten ts d ig e s te d in a steam bath fo r 9 to 10 h o u rs. A fter d ig e s t io n , th e tubes were cen tr ifu g e d and the s o lu b le m a ter ia l d is ­ carded . The a lk a l i in s o lu b le resid u e was washed tw ice w ith w ater, once w ith 95$ e th a n o l, and once w ith e th e r , in th a t o rd er. A fter the r e s id u a l e th e r had been removed in vacuo, 2 m l. o f 6 N h y d ro ch lo ric a cid were added to each resid u e and the necks o f the tubes se a le d o f f . The samples were then d ig e s te d fo r another 30 hours in the steam bath to hyd rolyze th e c h i t i n . The tubes were c o o led , opened, and placed in 29 a vacuum d e s ic c a to r over potassium hydroxide p e l l e t s and calcium c h lo r id e to remove as much o f the h y d ro ch lo ric acid as p o s s ib le . The con ten ts were f i n a l l y tr a n sfe rr e d to a volum etric f la s k , and a liq u o ts analysed by the D ische method fo r t h e ir hexosamine content . The jug ch i t i n were c a lc u la te d by m u ltip ly in g the juM GA by the m olecular w eight o f N -a c ety lg lu e o sa m in e . Melanin E stim a tio n .— The melanin co n ten t o f p la n ts at various ages was estim ated by d ig e s tin g 10 mg. sa m p le s.o f d ried p la n t m a ter ia l w ith 1 m l. o f 0 .9 N sodium h yd roxid e. The o p t ic a l d e n s ity o f the s l i g h t l y tu rb id s o lu tio n s obtained (d ilu te d .1:6) was measured a t 5 p o in ts between iiOO and 600 mju in th e spectrophotom eter, as recommended by S c h a e ffe r *(1993) • These d a ta , when p lo tte d as th e logarithm o f th e o p t ic a l d e n s ity a g a in st the wave le n g th , y ie ld e d th e s tr a ig h t li n e s lo p e s charact e r i s t i c o f the melanoid pigment s . T otal L ipids .--T he t o t a l l i p i d content o f fr e s h plan t m a teria l was determ ined by the method o f F olch , L ees, and S ta n le y (1 9 9 /) , fo r animal tis s u e s . To check on the e f f ic ie n c y o f th e chloroform -m ethanol e x tr a c ­ t io n w ith fu n gal m a teria l a- q u a n tity o f fr e s h B la st 0 c l a d i e l l a p la n t m a ter ia l (somewhat la r g e r than was used in the experim ents to be d escrib ed ) was e x tr a cte d by th e method o f F olch, _et a l . The e x tr a cte d r e sid u e was then reflu x ed fo r 20 hours w ith a fr e sh p o rtio n o f the chloroform -m ethanol so lv en t m ixture, and the q u a n tity of l i p i d in t h is second ex tra ct determ ined. When t h is was done i t was found that on ly 3 .2 $ o f th e t o t a l q x tra cta b le l i p i d remained a ft e r th e i n i t i a l e x tr a c tio n , 30 and th e method was th e r e fo r e considered a p p lic a b le io the a n a ly se s o f B la s t o c l a d ie lla m a t e r ia l. q u a n tity o f No attempt was made to correct fo r the sm all y -c a r o te n e which was e x tr a cte d along w ith the l i p i d s . P rep arative Procedures Dowex-50 R esin Columns.—Dowex-50 r e s in (x 8 , 100 - 200 mesh; Bio Rad L ab oratories; Reagent grade) was r e g u la r ly regenerated b efo re use w ith two com plete c y c le s o f acid and a l k a l i , w ith thorough d i s t i l l e d water washes between each trea tm en t. To o b ta in the r e s in in the hydrogen form, th e la s t treatm ent was w ith 2 N h y d ro ch lo ric a cid ; when the r e s in was required in the potassium form , th e l a s t treatm ent was w ith 2 N potassium h yd roxid e. F ollow ing reg en era tio n th e r e s in was washed e x h a u s tiv e ly w ith g la s s d i s t i l l e d w ater b efo re u s e . F r u c to se - 6- ph osp hate.—A f a i r l y crude sample from Schwarz labora­ t o r ie s was p u r ifie d by two p r e c ip ita tio n s as the a lc o h o l in s o lu b le , barium s a lt at pH 8 .0 . The rem aining y e llo w c o lo r was removed by tr e a tin g w ith N o rite at 8 0 °C .,‘ and the c o lo r le s s m a teria l converted to th e sodium or potassium s a l t by p r e c ip ita tin g the barium w ith sodium s u l f a t e , or by running th e s o lu tio n through a Dowex- 50 (K+ ) column, r e s p e c tiv e ly '. A barium s a l t from th e Sigma Chemical Co. gave c le a r , c o lo r le s s s o lu t io n s , and was converted to the potassium s a l t by the column pro­ ced u re. This m a te r ia l'a ssa y e d as 8 0 .6 $ F - 6 -P by the ant hr one method. Due to the fa c t that a l l samples of F - 6 -P on th e market a lso contained C-6 -P , a sample of pure F - 6 -P was prepared from com m ercially 3.1 a v a ila b le f r u c t o s e - 1 , 6 -d ip h osp h ate (FDP) . The FDP (Schwarz la b o r a to r ie s , I n c .) was r e c r y s t a lliz e d I4 tim es as the cyclohexylammonium s a lt by the method o f McOilvery 1,1953) • A fter r e c r y s t a l l iz a t io n , the s a lt was converted t o .t h e fr e e a cid by passage through a Dowex-50 (H ') column. A sam ple, chromatographed in propanol-ammonium h yd roxid e-w ater, was found to be f r e e o f hexose-unonophosphate e s t e r s and in organ ic phosphate. The p u r ifie d FDP was then hydrolyzed w ith 1 N hydrobromic a c id , and th e barium F - 6 -P s a l t is o la t e d by th e method o f Neuberg, L u s tig , and Rothenberg (19U3)* p h ate. It contained 9 0 .1 $ F- 6 -P and 0 .1 5 $ in o rg a n ic phos­ The barium s a l t was converted to the potassium d e r iv a tiv e by th e column method d escrib ed above. N -a cety lg lu co sa m in e.— This compound was prepared from d-glucosam ine h yd roch lorid e (C a lifo r n ia Foundation fo r B iochem ical Research) by the method o f Roseman and Ludowieg (195U )* D -glu eosam in e- 6 - ph osp hate.--A few m illigram s o f GA-6 -P were synth e­ s iz e d by th e-ch em ica l method o f Anderson and P e r c iv a l (1 9 5 6 ). In su f­ f i c i e n t m a teria l was is o la te d to allow chem ical c h a r a c te r iz a tio n , but th e product did have the same chromatographic p ro p erties as an a u th e n tic sample (prepared by the polyphosphoric acid method) which was very k in d ly provided by D r. Saul Roseman (D .istle r , Merrick, and Roseman, 1958) * "With both th e Hanes and Isherwood sp ray, and w ith n in h yd rin , th e s y n th e tic compound gave a s in g le spot w ith the same Rj> as R.oseman*s compound * 32 M iscellan eou s P r e p a r a tio n s■—T ricalcium -phosphate g e l was prepared accordin g to th e procedure o f K e ilin and Hartree ^1938). The product contain ed 17 mg. dry weight o f g e l per m l. o f su sp en sio n . S o lu tio n s o f protamine s u lf a t e (E li L ill y ) were prepared c o n ta in ­ in g 20 mg. per m l., and the pH was adjusted to 3 .8 w ith 1 N A c etic a c id . 3 -P h osp h oglyceric a cid (Schwarz Lab. I n c .) was r e c r y s t a lliz e d two tim es by the procedure o f Neuberg and L u stig (191+2),. T h e o r e tic a l r e c o v e r ie s f o r t o t a l organic phosphate were obtained w ith the product. l,2 ,]+ -a m in o -n a p h ,th o l-su lfo n ic acid (Eastman Kodak) fo r the F iske and Subbarow r e a c tio n was r e c r y s t a lliz e d by th e method o f the authors (1925) • The ammonium sulfam ate (Eastman Kodak, p r a c tic a l) fo r the D ische r e a c tio n was r e c r y s t a lliz e d from eth an ol-w ater m ix tu res. Paradim ethyl- aminobenzaldehyde was r e c r y s t a lliz e d by th e procedure given by Tracey (1955) • Sources o f Chemicals and B iochem icals AH o f the in organ ic chem icals used in the work reported here were o f reagent or comparable grad e. The in d o le , an th ron e, and a c e ty la c e to n e used were products of Eastman Kodak. The barium- and d ip o ta s siu m -s a lts o f G-6-P were obtained from th e Sigma Chemical Co. Ninhydrin was procured from e it h e r the Sigma Chemical Company or the N u tr itio n a l B iochem ical Corp. The 1- glutam ine was a product o f the C a lifo r n ia Foundation fo r B iochem ical R esearch, and the 1 - glutam ic acid o f the P fa n s tie h l Chemical Corp. ATI oth er amino acid s were ob tain ed from N u tr itio n a l B iochem icals Corp. 33 The g la s s beads used fo r hom ogenization in the Omni-mixer were obtained from th e M innesota Mining and Manufacturing Co. 3h EXPERIMENTAL The Free Amino Acid Pools ■in B la s t o c la d ie lla As a prelim in ary approach to th e problem o f n itr o g e n metabolism and i t s r e la t io n s h ip to d if f e r e n t ia t io n in B la s t o c la d ie lla i t appeared th a t a stu d y o f th e s o lu b le amino acid pools might w e ll provide some in d ic a tio n o f th e most p r o fita b le areas to i n v e s t i g a t e . Any obvious d iffe r e n c e s in th e se compounds between the two m orphological form s, e it h e r q u a lit a t iv e or q u a n tita tiv e , should r e s u lt from a lte r a tio n s in c e r ta in -b io s y n t h e tic sy stem s. I f th is were s o , changes observed in s p e c i f i c pools would h e lp to pin poin t th o se m etab olic pathways which d i f f e r fundam entally between the a lte r n a tiv e p lan t t y p e s . To check the above, mature R .S. and O.C. p la n ts derived from c u ltu r e s grown, fo r a period o f s e v e r a l g e n e ra tio n s, were h a rv ested , d r ie d , and t h e ir f r e e amino A cid s_e x tr a c te d . When th e se e x tr a c ts were chromatographed tw o -d im en sio n a lly , s t r ik in g d iffe r e n c e s were observed, both q u a n tita tiv e and q u a lit a t iv e (F ig . 2 ) . I t was' obvious even from v is u a l in s p e c tio n o f the' chromatograms that the q u a n titie s o f alm ost a l l th e f r e e amino a cid pools in th e R .S. p la n ts were g r e a tly decreased in comparison w ith the O.C. p la n ts . 'T h is was not tr u e , however, fo r glutam ic and a s p a r tic a c id s , and to a l e s s e r extent fo r two or th ree o f the unknown compounds f a l l i n g in th e same general area o f th e chroma­ tograms . e x tr a c ts . These a l l appeared to remain at the same l e v e l in both p la n t In a d d itio n to the quantit a tiv e changes, a new compound 35 O.C. LEUCINE PHENYL ALANINE TRYPTOPHANE VALINE TYROSINE O '" PROLINE ALANINE GLUTAMATE HYDROXYPROLINE ASPARTATE RGININI SERINE LYSINE HISTIDINE R.S. o LEUCINE VALINE TRYPTOPHANE TYROSINE ALANINE / UNIDENTIFIED THREONINE o GLUTAMATE 9 P GLYCINE 0 ARGININE , _ HISTIDINE 1 1 0 O SERINE ASPARTATE ^ ° V UNIDENTIFIED C O Figure 2 The Soluble Amino A cids o f O.C. and R .S. P la n ts An e x tr a c t o f 10 mg. dry w eight o f p la n t m a teria l chromatographed in phenol-w ater (h o r iz o n ta l) and b u ta n o l-p ro p io n ic acid -w ater ( v e r t i c a l ) . The amino a c id s were d e te c ted w ith ninhydrin. 36 appeared in P .S . e x tr a c ts below g ly c in e marked on the chromatogram as u n identified") which has been t e n t a t iv e ly id e n t if ie d as a sp a ra g in e. The unknown compound in the cen ter o f the tr ia n g le formed by s e r in e , glutam ate, and a sp a r ta te in the O.C. chromatogram, on the oth er hand, could not be d e te c ted in m a teria l from R .S. p la n ts . To serv e as a check on the v is u a l e s tim a te s , q u a n tita tiv e d e te r ­ m inations were undertaken. D u p lica te s e t s o f chromatograms fo r each plan t form were prepared and the glutam ic a c id , a s p a r tic a c id , and a few o th er w e ll sep arated amino a cid s estim ated by th e method o f Landua and Awapara (I9lt9; Awapara, 19U 9 )• The r e s u lt s of th e s e a n a ly ses are given in Table I . TABLE I ANALYSIS OF THE SOLUBLE AMINO ACID P00I£ IN 0 .0 . AND R .S. PLANTS OF BLASTOCLADIELLA . pM Amino Acid per Gram Dry Weight Amino Acid pg Amino Acid-N per Gram Dry Weight O.C. R. S. O.C . R. S. Glut amat e 29 .2 30.2 It08.8 U22.8 A spartate 1 1 .8 13 .2 165-2 lblt - 8 . 52 .H 5-1 733 -6 7 1 .6 L ysine 16 .h 8 .8 U59-2 2I4.6 .It A rginine - lit-It 7-6 806 .It U25.6 1 5 .6 9 .6 2 l8 .lt 13 It .It llt.lt ca.O 201.6 ca .0 2993 .2 1U85-6 A lanine Threonine Tyrosine T otal . 3' These d ata corroborated the e stim a te s fo r glutam ic and a sp a r tic a cid s . In f a c t , the co n cen tra tio n s of both compounds in crea sed s l i g h t l y in th e P . S . m a te r ia l. At the same tim e the t o t a l amino a cid n itro g e n (as rep resen ted by the fr a c t io n analyzed) decreased by 50$ in th e R .S. When th e change was c a lc u la te d fo r a l l amino a cid s oth er than glutam ic and a s p a r t ic , th e drop amounted to 61$, a ra th er d r a s tic change. A lanine and ty r o s in e showed th e m ost-severe d e c r e a s e s , but i t was - obvious from v is u a l in s p e c tio n th at oth er amino a cid s (which could not i be measured because o f t h e ir overlap pin g p o s itio n s ) underwent changes o f a s im ila r m agnitude. The r e te n tio n o f the glutam ic acid pool in th e R .S. p la n ts did su g g est that i t might indeed be worth in v e s tig a tin g fu r th e r . This seemed p a r tic u la r ly p e r tin e n t s in c e glutam ate might have served as the lin k ( e . g . , by tran sam in ation , or the glutam ic dehydrogenase system ) between the p u ta tiv e lo cu s o f the bicarbonate e f f e c t , a -k e to g lu ta r a te , and th e s y n th e tic pathway fo r c h it in s y n th e s is . The p o s itio n o f glu ­ tam ate in th e l a t t e r w i l l be made evid en t in th e fo llo w in g s e c t io n s . Glucosamine S yn th etase in P lant E xtracts The s te p in the b io s y n th e sis o f c h it in in c lo s e s t proxim ity to . glutam ic acid i s th at cata ly zed by the enzyme glucosam ine sy n th eta se: H exose-6-phosphate + 1 - glutam ine — > 1-G lutam ate. Glueosamine-6 - phosphate + T h erefore, th is enzyme was assayed in R .S. and O.C. p la n ts to se e i f th er e was any c o r r e la tio n between i t s a c t i v i t y and the l e v e l o f the fr e e glutam ic acid p o o l. It was f i r s t n ecessa ry to e s t a b lis h th a t the r e a c tio n occurred w ith glutam ine as a s u b s tr a te , rather than 33 F- 6 -P and ammonia. For t h is purpose, a m a ltip le -g e n e r a tio n c u ltu r e o f 0 .C . p la n ts was used s in c e such m a teria l was much sim p ler to grow and homogenize than R .S. p la n ts . Two grams wet weight o f a 7 day old c u l_ 2 tu re were ground in a g la ss homogenizer w ith 10 m l. 6-7 x 10 phosphate b u ffe r , pH 6 . 8 . M. F iv e -te n th s ml. o f the whole homogenate, 30 juM glutam ine, 30 jaM 0 -6 -P , and 3U>iM phosphate b u ffe r , pH 6 . 8 , in a t o t a l volume o f 3*0 m l., were incubated at 32 °C. fo r z e r o , o n e -h a lf, and 2 h o u rs. p e r io d s! The fo llo w in g c o n tr o ls were incubated fo r th e same tim e com plete system (a) w ith b o ile d homogenate, (b) minus homo­ g en a te, (c ) minus glutam ine, and (d) minus G-6 - P . The r e a c tio n m ixtures were in a c tiv a te d and d e p r o te in ize d at th e in d ica ted tim es w ith 0 . 3 m l. o f 35% TCA. The com plete r e a c tio n m ixtures contained a compound w ith low m o b ility 0 . 1 in b u tan ol-p ro p io n ic a cid -w a ter) which reacted w ith both th e ninhydrin and Hanes.and Isherwood sp r a y s. This s p o t, presum­ ab ly GA-6 -P , showed a p rop ortio n a l in c r ea se w ith the time o f in cu b a tio n and was absent from a l l the c o n tr o l m ix tu r es. In a d d itio n , in cu b a tio n o f th e whole homogenate plus s u b s tr a te s , in the absence o f phosphate b u ffer le d to the production of a co n sid era b le q u a n tity o f fr e e GA. This appeared to be due to phosphatase a c tio n upon the GA-6 -P . These r e s u lt s in d ic a te d th at the sy n th eta se enzyme t a ) was present in B ia s t o c l a d ie lla and (b) u t i l i z e d glutamine as the source o f amino groups in th e s y n th e s is o f GA-6-P . To e s t a b lis h the lo c a tio n o f the enzyme in th e extract., th e a c t i v it y in whole homogenates was compared w ith that r eta in e d -.in supernatants a f t e r c e n tr ifu g a tio n at 300 - and 39 22,000 x g . at 0 t o [|° C . A ll th e se fr a c tio n s appeared to have f u l l y comparable a c t i v i t y as estim ated by the s iz e o f the low m o b ility (Rf 0 .1 6 , propanol-ammonia) ninhydrin p o s it iv e compound. This seemed to e s t a b lis h th e '‘s o lu b ility '' o f the enzyme, and a l l fu r th e r experim ents were undertaken w ith th e 22,000 x g . su p ern a ta n ts. In the same experiment i t was found that- F-6-P had an. a c t i v i t y equal to that o f 0-6-P in the sy n th eta se r e a c tio n . F r u c to se -6 - phosphate was th e r e fo r e used in subsequent experim ents because i t was r e a d ily a v a ila b le , l e s s e x p e n siv e, and according to Blum enthal, e t a l . ( 195 5 )* the p referred su b str a te fo r th e r e a c tio n . The Product o f Glucosamine S yn th etase A c t i v i t y .— To e s t a b lis h w ith reason ab le c e r ta in ty that the r e a c tio n product was in fa c t GA-6-P, a p a r t ia l p u r if ic a tio n was undertaken (F ig . 3) • The barium p r e c ip ita tio n s were th o se c o n v e n tio n a lly used ((Jmbreit., B u r ris, and S ta u fe r , 1957) to sep a ra te m ixtures o f phosphate e s t e r s . F r u c to se -6 - phosphate and GA-6-P were th e only compounds expected in the barium so lu b le a lc o h o l in s o lu b le fr a c tio n ^BSAI'), but when t h is m a teria l was r e d isso lv e d and chromatographed both glutam ine and glutam ic a cid were s t i l l present as contam inants. The r e s u lt was somewhat .su r p r isin g s in c e L e lo ir and C ardini (1953) had used th e .procedure to sep a ra te G-6-P and GA-6-P from id e n t ic a l m ixtures and did not report the presence o f amino a c id s , On recourse ,to the lit e r a t u r e i t was found (Foreman, 191U) that- glutam ic acid does pre­ c i p i t a t e as an a lc o h o l-in s o lu b le barium or calcium s a lt . To ob v ia te th e p r e c ip ita tio n problem w ith amino a cid s the m a te r ia l, alread y tw ice p r e c ip ita te d as the BSAI s a l t s , was run in to a Dowex-50 Uo Figure 3 GLUCOSAMINE-6 -PHOSPHATE PURIFICATION PROCEDURE REACTION MLXTUBE CONTAINING TCA (20 .5 m l.) I C entrifuged > P r o te in d iscard ed SUPERNATANT - I E xtracted w ith k volumes e th y l eth er to remove TCA. The pH adjusted to 8 .2 w ith KOH and p h en o lp h th a lein . l.H m l. 25 $ . barium a c e ta te added w ith m ixing. I Centrifuged' P r e c ip ita te discard ed BARIUM SOLUBLE SUPERNATANT place at - l 8 °C . fo r 2 . 5 h r . I4 volumes o f 95 $ eth a n o l added, placed C entrifuged Supernatant discard ed BARIUM-SOLUBLE-ALCOHOL-INSOLUBLE PRECIPITATE lBSAI-1) ' Washed 3 tim es w ith 3 m l. 95$ e th a n o l. R ed issolved in 2 .5 m l. 1 0 - XN HC1, 2 X 10_2N H2S04 added to p r e c ip ita te BaS04 . C entrifuged ^BaS04 discard ed SUPERNATANT - II A lcohol p r e c ip ita tio n as the BSAI s a l t r e p e a te d . ■BSAI. - 2 R .edissolved in a minimal volume o f d ilu t e HC1 and the barium removed w ith Na 2S04 , and c e n tr ifu g a tio n . I ^ S o lu tio n run in to a.Dowex~50 (H ) column (0 .8 X 25 cm.) and a s e r ie s o f 10 ml fr a c tio n s elu ted w ith w a ter. F ra ctio n 1 2 3 -8 9-20 i ^ Content F- 6-P , in o rg a n ic phosphate N egative GA-6-P N egative ^ h e s e r e s u lt s were obtained by chromatography and de­ t e c t io n w ith ninhydrin and Hanes and I sherwood sp ra y s. hi 1^1 . (H ) column and a s e r ie s o f fr a c tio n s e lu te d w ith w ater KW olf, M orita, and Nakada, 19^ 6). The r e s u lt s are l i s t e d at the bottom o f F ig . 3 , and show good sep a ra tio n o f the GA-6-P from the oth er compounds p resen t in th e r e a c tio n m ix tu res. I t was subsequently found th a t r e a c tio n m ixtures which had been d e p r o te in ize d w ith TCA, .cou.ld be d ir e c t ly sep arated by the same column procedure w ithout e th e r e x tr a c tio n to remove th e TCA, w ith e q u a lly s a t is f a c t o r y s e p a r a tio n s . The amino a cid s which remained on the column _i were recovered when d e s ir e d , by e lu tio n w ith 2 x 10 N ammonium h y d ro x id e. The GA-6-P fr a c tio n s from the column were pooled and concen trated to a sm all volume in a f la s h evaporator at 30 to 35>°C. When t h is m a ter ia l was co-chromatographed w ith an a u th en tic sample prepared by D r. S au l Roseman, the two compounds had id e n t ic a l m o b ilitie s (as determ ined by both ninhydrin and Hanes and Isherwood sp rays) in th ree d if f e r e n t s o lv e n ts ; b u tan o l-p ro p io n ic a cid -w a ter, propanol-ammonium h yd roxid e-w ater, and ethanol-ammonium a c e ta te ( c . f . Appendix I I I fo r v a lu e s ) . In an attempt to i s o l a t e and fu r th e r p u r ify the e n zy m a tica lly generated GA-6-P, the pooled sample was tr e a ted w ith barium, th e BSAI - 1 s a l t is o la t e d , d is s o lv e d in 10 N h yd roch loric a c id , and fr e ed o f barium by the a d d itio n o f sodium s u l f a t e . This m a teria l was analyzed fo r i t s organic n itr o g e n and phosphorus c o n te n t, along w ith the sample provided by Dr. Roseman. It was found to co n ta in a ph osp horus/nitrogen r a tio o f l / l . 2 , as compared to l / 0 .9 * f o r Roseman1s m a te r ia l. When the same GA-6 -P (th a t had been r e -p r e c ip ita te d at pH 8 .2 ) was again chroma­ tographed, a second s-pot w ith an o f 0 .2 6 (propanol-ammonia) was found which had been absent from th e o r ig in a l column e lu a t e s . This m a ter ia l reduced a lk a lin e potassium permanganate and contained organic phosphorus, but d id not rea ct w ith n in h yd rin . I t was concluded that t h is was a decom position product from GA-6 -P produced during the a lk a lin e p r e c ip ita tio n s in c e i t could be removed w ith Dowex-50 (H*) . The im p u rity , which was not F- 6 -P , appeared in the f i r s t fr a c tio n from th e column. I t s spectrum had a peak at 2/3 mp, which agreed w ith Brown1 s report ( 195 .1 ) o f an in c r e a se in ab sorption at that wave le n g th when GA-6 -P s o lu tio n s were sto red at pH 8 .0 . The decom position did not occur when.GA-6 - P was sto red in the r e fr ig e r a to r at low pH. I t i s pre­ sumed that t h is decom position was th e cause o f the s l i g h t l y high n itro g e n v a lu es obtained in the a n a ly s e s, s in c e the r a tio should th eo­ r e t i c a l l y have been one. The spectrum o f the colo red product obtained in the D ische r ea c tio n w ith B la s t o c l a d ie lla GA-6 -P was id e n t ic a l to th a t o f GA. For both com­ pounds th e ab sorp tion decreased p r e c ip ito u s ly on e ith e r s id e o f a sharp maximum at U92 mp. In c o n c lu sio n , i t was decided th a t the product o f our enzyme r ea c tio n was GA-6-P. P u r ific a tio n o f Glucosamine S yn th etase ' The experim ents d escrib ed in the preceding paragraphs e s ta b lis h e d th e p resence o f glucosam ine sy n th eta se in c e l l f r e e preparations of B la s t o c la d ie ll a , and the su b str a te and products o f th e r e a c tio n . h3 However, b e fo r e b egin n in g a study o f th e r o le o f the enzyme during development i t was e s s e n t ia l to e s t a b lis h the optim al c o n d itio n s fo r it s a c tiv ity . To do t h is a p a r tia l p u r if ic a tio n o f the enzyme was attempt e d . P u r ific a tio n P roced ures.— A v a r ie t y o f co n v en tio n a l methods fo r enzyme p u r if ic a t io n were t r i e d . However, the great m ajority o f th ese r e s u lte d not on ly in a la c k o f p u r if ic a tio n but a ctu a l in a c tiv a tio n of th e ap p aren tly very l a b i l e enzyme. Some o f the more p ertin en t t r e a t ­ ments and t h e ir e f f e c t upon sy n th eta se a c t i v i t y are l i s t e d in Appendix IV. The enzyme l o s t 10 to 20$ o f i t s a c t i v i t y on sta n d in g at 0° C. fo r s e v e r a l hou rs, and from 56 to 80 $ on 12 hours d i a l y s i s ; both e f f e c t s -3 were a c c e le r a te d by the presence o f 10 M V ersen e. The a d d itio n -o f con cen trated d ia l y s a t e , or magnesium plus pyridoxal phosphate f a i l e d to r e s to r e the a c t i v i t y l o s t upon d i a l y s i s . _4 Both- g lu ta th io n e ('10 M) and - -4 F - 6 -P (9 x 10 M) had some s t a b i l i z i n g e f f e c t , but not enough to prevent s ig n if ic a n t lo s s e s in a c t i v i t y on sta n d in g , or upon d i a l y s i s . The a c t i v i t y was heat s e n s it iv e and, to a l e s s e r e x te n t, pH s e n s i t i v e . A ll attem pts to i s o l a t e th e enzyme by ammonium s u lf a t e fr a c tio n a tio n met w ith f a i l u r e . Although th e g r e a te st a c t i v i t y was always found in the f r a c t io n c o lle c t e d between 30 and h$% sa tu r a tio n w ith ammonium s u l f a t e , the s p e c if ic a c t i v i t y and/or t o t a l y ie ld were in every case reduced by th e treatm en t. I f , in a d d itio n , the r e d iss o lv e d ammonium s u lf a t e 'f r a c t io n was d ia liz e d a g a in st phosphate b u ffe r (10 a fu r th e r lo s s o f (%% in a c t i v i t y occu rred . - L M, pH 7.0'), The most s a t is f a c t o r y an d .rep ro d u cib le p u r if ic a tio n was obtained by tr e a t in g h igh speed su p ern a ta n ts, c o n ta in in g no phosphate b u ffe r , w ith a protamine s u lf a t e s o lu tio n adjusted to pH 5*8 (0 .1 7 mg. protamine s u lfa t e /m g .'p r o te in ) . Under th e s e c o n d itio n s the glucosam ine sy n th eta se a c t i v i t y remained in th e su p ern a ta n t, and p u r if ic a tio n s ranging from 3 .2 to 6.1*-fold w ith n e a r ly 100$ recovery were obtained ( c . f . Appendix IV) . ’ The enzyme was e f f e c t i v e l y adsorbed from th e protamine supernatant on tr ic a lc iu m phosphate g e l at pH £ . 6 . Attempts to e lu te th e enzyme w ith a s e r ie s o f phosphate b u ffe r s o f in c r e a s in g m o la r ity , or in c r e a s­ in g pH, or b o th , f a i l e d to give adequate r e c o v e r ie s in any one f r a c t io n . _ i However, in such s e r ie s 10 M phosphate b u ffer a t pH 7-0 always gave th e b e st r e s u l t s , and s in g le e x tr a c tio n s of t h is so rt did r e s u lt in some p u r if ic a t io n . Heppel and Hilmoe (19^1) reported th a t the enzyme, inorganic pyrophosphatase, could be e f f i c i e n t l y e lu te d from g e l preparations by i t s own s u b s tr a te , in organ ic pyrophosphate. T herefore, a s im ila r approach was t r ie d w ith the sy n th eta se enzyme from B la s t o c la d ie lla _3 u sin g 5 X 10 M F- 6 -P as th e e lu tin g a g e n t. The r e s u lt s were encourag­ in g , and le d to th e t r i a l o f h ig h er co n cen tra tio n s o f both the commercial and s y n th e tic F - 6 -P , as w e ll as G-6-P'. Table IT. The r e s u lt s are shown in ' I t was q u ite evid en t from th e se data th a t the commerical F- 6 -P was su p erio r to e it h e r the s y n th e tic F - 6 -P or the G-6 -P as an e lu tin g agent . The reduced e f f ic ie n c y o f the s y n th e tic F- 6 -P e lu tio n remains hS TABLE II GEL ELUTION WITH HEXOSE-PHOSPHATES a E lu tin g E ster S p e c if ic A c t iv it y With Protamine Supernatant S p e c if ic A ctiv ­ Percentage Increase i t y With Gel in S p e c if ic Act i v i t y E luate 0- 6 - P 8.59 16.9 97 F - 6 -P (s y n th .) 9 -hi 12 .6 3k F - 6 -P (Sigma) 9 -66 20 .5 112 Conditions*. Equal q u a n titie s o f th e g e l (prepared by ad so rp tio n from th e protamine supernatan t) were e lu te d w ith 3 ml- (1 0 " 2 M) o f th e in d ic a te d e s t e r . The e lu a te s were assayed w ith 10 • juM o f the same hexose-p h osp h ate, 20 pM glutam ine, and lj.0 pM phosphate b u ffe r , gH 6-5* in a f i n a l volume o f 2 .1 m l., fo r 1 hour ; T = 3 0 .6 C . The r e a c tio n was stopped w ith '(% Na2W04 (0 .0 1 m l.) and 1 N HC1 (0 .0 6 m l.) , and the GA-6 -P determ ined by th e D ische method. S p e c if ic A c tiv ity : pM GA-6 -P/m g. p r o te in /h r . ^Refers to the s p e c if ic a c t iv it y w ith the same e s t e r as used fo r e lu t io n . a p s z z le s in c e i t had been as e f f e c t i v e as the commercial F - 6 -P (and su p e r io r to 0- 6 - P) in th e glucosam ine sy n th eta se r ea c tio n w ith the protamine su p e r n a ta n t. The pH Optimum o f Glucosamine S y n th e ta s e .—An e a r ly d eterm in ation o f the pH optimum w ith F - 6 -P as the su b str a te had in d ic a te d a broad range o f almost equal a c t i v i t y from pH 3*5 to j . 0 , However, in the l i g h t o f the a c t i v i t y demonstrated by G-6 -P , the pH respon se fo r both compounds was examined at, c lo s e r in te r v a ls (from pH 5 .0 to 8 .5) u sin g the h ig h speed su p ern a ta n ts. 6 .5 to 6 .7 . The pH optimum (F ig . 5) occurred at pH -An in t e r p r e ta tio n o f th e d iffe r e n c e s in th e shape o f the c u r v e s, and t h e ir .inverse r e la t io n s h ip at h ig h er pH1s i s found in the d is c u s s io n . Due to the presence o f F - 6 -P in the enzyme p reparation p i r i f i e d w ith t h is e s t e r , the-pH response could only be determined u sin g i t as s u b s tr a te . The r e s u lt s were e s s e n t i a l l y th e same as th o se shown in Figure £ . Because o f the extreme l a b i l i t y o f the enzyme, fu rth e r attem pts a t p u r if ic a t io n were abandoned. The procedure which y ie ld e d th e maximal f i n a l p u r if ic a t io n i s shown in Figure I4 and th e data obtained in Table I I I . Time Course o f th e Glucosamine S yn th etase R e a ctio n .—U t i li z i n g th e enzyme p u r ifie d in th e above manner and both F - 6 -P and G-6 -P as sub­ s t r a t e s , the r e la t io n s h ip between GA-6 -P form ation and in cu b a tio n tim e was e s ta b lis h e d (F ig . 6 ) . The la g obtained w ith G-6 -P and i t s absence w ith F - 6 -P , was o f .importance in e s t a b lis h in g which o f the two served as th e primary su b str a te fo r th e r e a c tio n (s e e l a t e r d is c u s s io n ) . The l i n e a r i t y o f the response curve w ith F - 6 -P up to 30 minutes in d ic a te d that any tim e during t h is in t e r v a l would be s a t is f a c t o r y fo r a ssa y in g th e enzyme in plan t hom ogenates. Glucosamine Production v s . Enzyme Concent rat i o n .— The q u a n tity of GA-6 -P produced from F- 6 -P by the p u r ifie d enzyme was a lin e a r fu n c tio n o f th e p r o te in co n cen tra tio n from 0 up to approxim ately 0 . 8 mg. o f p r o te in per tube (F ig . 7 )• S u b stra te S p e c i f i c i t y .— A s e r ie s o f c o n tr o ls s e t 'u p to e s t a b lis h (a.) th e s p e c i f i c i t y o f the p a r t ia l ly p u r ifie d enzyme, and ^b) the absence o f any non-enzym atic a c t i v i t y , are ta b u la ted in Table EV. Figure k> Glucoasamine S yn th eta se P u r if ic a t io n Procedure. The r e s u lt s obtained by t h is procedure are given in Table ITT. The tem perature was m aintained between 0 and i| C. throughout th e p u r if ic a t io n . The g e l’ su sp en sio n s were s tir r e d in t e r ­ m it te n tly by hand because m echanical s t i r r i n g c o n s is t e n t ly reduced th e r e c o v e r ie s obtained . A ll c e n tr ifu g a tio n s but •the f i r s t were fo r 5 minutes at lU ,300 x g . Figure 5 . The R e la tio n Between Glucosamine S yn th etase A c tiv ity and pH. Two-tenths m l. o f a 22,000 x g . supernatant (6 .3 mg. p r o te in per m l.) were incubated w ith 20 pM F-6-P (o r G -6-P), 20 pM glutam ine, and 100 pM phosphate b u ffe r , in a f i n a l volume o f 2 .0 m l., f o r 20 min. a t 3 0 .6 C. The r e a c tio n was stopped by th e a d d itio n o f 0 .0 1 m l. 7% Na2W04 and 0 .08 ml. 1 N HC1. The GA-6-P was determ ined by the D ische method, and a l l v a lu e s co rrected fo r t h e ir unincubated c o n tr o ls . F igure 6 Time Course o f th e Glucosamine S yn thetase R ea ctio n . The enzyme (O.lj mg.) e lu te d from tr ic a lc iu m phosphate g e l w ith 10" 1 M, pH 7 -0 , phosphate b u ffe r was incubated and assayed under th e c o n d itio n s given in F ig . 3, excep t that the pH was 6 .3 and the tim e o f in cu b a tio n v a r ie d . Figure 7 The E ffe c t o f Glucosamine S yn th etase C oncentration on the R eaction R ate. _ i The enzyme e lu te d from tr ic a lc iu m phosphate g e l w ith 10 M, pH 7 .0 , phosphate b u ffe r was incubated w ith 20 pM F -6-P , 20 pM glutam ine, 100 pM phosphate b u ffe r , pH J . 0 , in a f i n a l v o l . o f 2 .0 m l., fo r 20 min. at 3 0 .6 ° C. See F ig . 3 fo r oth er d e t a i l s . U8 Figure Ij. GLUCOSAMINE SYNTHETASE PURIFICATION PROCEDURE 0 .C'. p lan t m a ter ia l homogenized 3 min. in th e Omni-mixer (1 gm. wet wt . to U gm. g la s s beads and U m l. g la s s d i s t i l l e d w a te r ). I Centrifugecj 30 min. at. 22000x g I Sediment and beads discard ed SUPERNATANT Protamine s u lf a t e (20 m g./m l. pH 5 -8 ) added slo w ly w ith s t ir r in g at th e r a te o f .O .iy mg. per-mg. o f p r o te in , and the mixture allow ed to stand 10 min . C entrifuged Sediment discarded PROTAMINE SUPERNATANT Sodium A cetate b u ffe r (1 M, pH 5*6) added slo w ly w ith s t ir r in g to a f i n a l c o n c en tr a tio n o f .10"2 M. I Calcium phosphate g e l added to the protamine supernatant (1 .2 mg. g e l / mg. p r o te in ) s t ir r e d fo r 15 min. i C entrifuged Supernatant discard ed T GEL S t ir r e d fo r 15 m in. w ith 5 m l. 10"2 M F -6 -P /2 1 .6 mg. g e l. GEL S tir r e d fo r 15 min. w ith 5 m l. 10“ * M phosphate b u ffe r , pH 7 -0 /2 1 .6 mg. g e l. Centrifuge'd. C entrifuged FIRST F-6-P ELUATE GEL S tir r e d f o r 15 min. w ith 5 m l. 10"2 M F -6 -P /2 1 .6 mg. g e l. SECOND F-6-P ELUATE FIRST PHOSPHATE ELUATE GEL S tir r e d fo r 15 min. w ith 5 m l. 10"* M phosphate b u ffe r pH 7*0/21.6 mg. g e l. SECOND PHOSPHATE ELUATE *9 O j jJ •H o o csi CL Ll 50 TABLE r r i GLUCOSAMINE SYNTHETASE PURIFICATION P r o te in Cone., mg ./m l. F ra ctio n T otal P ro tein T otal U nits o f A c tiv ity S p e c ific A c tiv ity 3-79 P u r if i­ c a tio n Supernatant 5.1 5 56.8 215 Protamine Supernatant 1.6 1 8 .5 236 12.8 A djusted to pH 5.9 1.6 18 .5 209 1,1.3 F ir s t F - 6 -P e lu a te 0 .2 5 ' 1.2k 67 53 .6 l U . l fo ld Second F - 6 -P e lu a te 0 .07 0 .3 5 25 7 2 .5 1 9 .1 fo ld F ir s t Phosphate e lu a te 0.72 ■ 3-6 52 17 .1 U -5 fo ld Second Phosphate e lu a te 0 .0 8 0.38 k 11.3 C on d ition s: . 3 -U fo ld Each enzyme f r a c t io n was incubated fo r 1 hour w ith 30 pM glutam ine, 30 pM F -6-P , 60 pM phosphate b u ffe r , pH 7^0, and w ater to g iv e a f i n a l volume o f 3*0 mlj T’= 30*3 C. The r e a c tio n was stopped w ith Tungstate-H Cl, and th e GA-6-P det ermined by th e D ische method. TABLE IV CONTROL REACTION MIXTURES INACTIVE WITH THE PARTIALLY PURIFIED ENZYME S u b stra tes Enzyme Phosphate B u ffer pH 6 .5 Volume 0 . 2 5 m l. 20 pM glutam ine 100 pM 2 .0 ml. 0 . 2 5 m l. 20 pM F-6-P 100 pM 2 .0 m l. 0 .2 5 m l. 20 pM 0,-6-P 100 pM 2 .0 m l. 0 .25 m l. 20 pM glutam ate, 20 pM F-6-P 100 pM - 2 .0 ml. 0 . 2 5 m l. 20 pM a sp a r a g in e ,20 pM F-6-P 100 pM 2 .0 ml. 0 .2 5 m l. 10 pM NH4C1, 10 pM F -6 -P , 10 pM ATP 70 pM 1 .1 m l. 0 .25 ml .(b o ile d ) 20 pM F -6-P , 20 pM glutam ine 100 pM 2 .0 m l. C on d ition s; The enzyme was a phosphate e lu a te (0 .3 b or 0 . 5 l mg. p r o te in / m l .) . Each r e a c tio n m ixture was incubated fo r 20 min. at 30 .6°C . , and inclu ded an unincubated c o n tr o l. The enzyme was in a c tiv a te d and th e p ro tein s.d en a tu red w ith 10% Na2W04 (0 .0 1 m l.) and 1 N HC1 (0.01+ m l.) , or by h e a tin g a t 100°C . fo r 5 min. CA-6-P was determ ined by the D ische method. From th e n e g a tiv e r e s u lt s obtained w ith th e s e m ixtures i t was con­ cluded th a t th e enzyme could o n ly u t i l i z e glutam ine as the aminon itr o g e n donor, and F-6-P or G-6-P as th e n itr o g e n a ccep tors in the r e a c t io n . S to ich io m e tr y o f th e Glucosamine Syn th etase R e a ctio n .—The reco v er­ i e s obtained fo r both r e a c ta n ts and p rod u cts, e x c lu s iv e o f th e h ex o sephosphates (s e e b elo w ), are shown in Table V. The r e s u lt s show a s a t is f a c t o r y , though not id e a l, r e la t io n s h ip between th e disappearance o f glutam ine and th e appearance o f GA-6-P, GA, and glutam ic a c id . In th e d is c u s s io n o f th e e a r ly experim ents ( c . f . p . 3 8 ) i t was noted th a t f r e e ( i . e . , non-phosphorylated) GA appeared in r e a c tio n m ixtures con­ t a in in g no in o r g a n ic phosphate. Upon in cu b a tin g the ammonium s u l f a t e , f r a c t io n (Exp. 31> Table V) w ith sodium c it r a t e b u ffe r in ste a d o f phosphate b u ffe r , 2 )4 . 5 % o f th e t o t a l product appeared as fr e e GA. However, in no ca se d id GA appear when in o rg a n ic phosphate was p resen t in th e r e a c tio n m ix tu r e s. I t i s obvious from th e la r g e in c r ea se in in ­ organ ic phosphate th a t a co n sid era b le q u a n tity o f the F-6-P was a lso l o s t by phosphatase a c tio n . Whether the deph osp horylation o f both GA-6-P and F-6-P was th e r e s u lt o f a s in g le n o n -s p e c ific phosphatase or two s p e c i f i c enzymes i s a moot p o in t. Of in t e r e s t in t h is r esp e c t i s the f a c t th a t Blum enthal, H em erline, and Roseman (.1956) is o la t e d a phosphatase from Neuro'spora cra ss a which was 25 tim es more a c tiv e toward GA-6-P than i t was toward oth er h e x o se-p h o sp h a tes. The optim al pH range o f 6 .0 to / . 5 fo r th e p u r ifie d glucosam ine phosphatase o f B lum enthal, e t a l . , overlap s th at o f th e glucosamine sy n th eta se and 32 TABLE V STOICHIOMETRY OF THE GLUCOSAMINE SYNTHETASE REACTION a GA-6-P Exp. 30 + U.6 Exp. 31 + 6 .8 GA + 2 .2 jiM During Incubation Glutamate Glutamine + 5.0 - k .8 + 9-9 - 7 .1 Inorganic Phosphate Exp. 65 Crude supernatant + .1 .8 + 2 .3 - 1 .6 Protamine s te p + 1 ,6 + 2 .2 - 2 .2 Ge.l e lu a te (,0.1 M phosphate) + 5 .0 + 5-0 - 5 -6 C on d ition s; Exp. 3 0 » A' supernatant d ia ly ze d 12 h r . at 0-2°C . v s . 10“ 2 M phosphate b u ffe r , pH 6-9 * 0 .^ m l. enzyme, 30 juM F -6-P , 30 juM glutam ine, 50 juM phosphate b u ffe r , pH 6 .9 , incubated fo r 2 h r . in a f i n a l volume o f 3*0 m l.; T = 31 C. Exp. 3 1 ■ The enzyme was a 30-50$ ammonium s u lf a t e fr a c tio n prepared from a supernatant d ia ly z e d v s . 10" 2M phosphate b u ffe r , pH 6 .9 , fo r 12 h r . a t 0-2 C ., and r e d iss o lv e d in 10"J-M sodium c it r a t e b u ffe r , pH 6 .8 . The in cu b a tio n con­ d it io n s were the same as those, in Exp. 3 0 , except that sodium c it r a t e was s u b s titu te d fo r the phosphate b u ffe r . Exp. 6 5 . These'were enzyme fr a c tio n s obtained by the pro­ cedure given in F ig . 5* Each f r a c t io n was incubated w ith 20 juM glutam ine, 20 juM F -6-P , and 100 jiM phosphate b u ffe r , pH 6 -5 , fo r 20 min. in a f i n a l volume of 2 .0 m l.; T = 30 .6°C . R eactants and products were analyzed by th e D ische method fo r g lu c o s- am ines, and th e method o f Kay, e t a l . fo r the- amino a c id s . A ll v a lu e s were co rrected fo r t h e ir unincubated c o n t r o ls , 53 such an enzyme could co n ceiv a b ly be present and a c tiv e in B la s t o c la d ie lla homogenates . Attempts to e s t a b lis h a 1 :1 r a t io between th e disappearance o f F -6-P '^or G-6-P) and th e appearance o f GA-6-P were only m oderately s u c c e s s f u l, even w ith th e p a r t ia l ly p u r ifie d enzyme. Table VI l i s t s th e r e s u lt s o f a n alyses fo r changes in the sugar phosphates during a 30 minute in c u b a tio n o f th e enzyme w ith F-6-P and G-6-P as th e sub­ str a te s . The d a ta s tr o n g ly su ggested the presence o f co n sid era b le phosphoglucose isom erase a c t i v i t y in th e p a r t ia l ly p u r ifie d enzyme. To v e r if y t h i s , th e c o n tr o l tubes co n ta in in g o n ly F-6-P were analyzed fo r th e presence o f G-6-P b efo re and a f t e r in cu b a tio n by measuring th e r a te s of TPN red u ctio n w ith g lu c o s e -6 - phosphate dehydrogenase (S ig m a ).The r e s u lt s are given in Table V II . TABLE VT HEXOSE-PHOSPHAIE CONVERSION'DURING INCUBATION WITH THE PARTIALLY PURIFIED GLUCOSAMINE. SYNTHETASE A juM During Incubation ' S u b stra te GA-6-P F-6-P G-6-P F-6-P + 1 .1 - U.0 + 2 .5 G-6-P + 0 .5 + 2*9’ - U-5 C on d ition s; The p a r t ia l ly p u r ifie d enzyme and r e a c tio n m ixtures were as in d ic a te d fo-r F ig . 6 . The tim e o f in cu b a tio n was 30 min,. TABLE vrr GLUCOSE-6-PHOSPHATE DEHYDROGENASE ■ASSAY FOR ISOMERASE ACTIVITY IN THE PARTIALLY PURIFIED ENZYME Time in Minutes O p tica l D e n sity C ontrol Experim ental 2 ' ' 0.015 O.OU2 U 0 .02U 0 .063 6 0.032 0 .0 7 1 8 O.O37 . 0.087 10 o.oi +5 0 .09I1 15 0 .061 0 .112 -25 0 .095 0.11*6 C on d itio n s: To d e te c t phosphoglucose isom erase a c t i v i t y in the phos­ phate g e l- e lu a t e , 0 . 2 5 ml* o f the enzyme (O.I4 mg. p r o te in ) were incubated w ith 20 juM F-6-P and 100 pM phosphate b u ffe r , pH 6*5jr in a volume o f 2 . 0 . m l. f o r 20 m in ., and th en heat in a c tiv a te d . An unincubated c o n tr o l was prepared in th e same fa s h io n w ith enzyme b o ile d b efo re a d d itio n . For th e G-6-P a ssa y , 1 .2 m l. 10- 1 M phosphate, pH 7 *5 , 0 .3 m l. 10“ 1 M MgCl2, 0 .1 m l. 2 .5 x 10"3M TPN, 0 .1 ml. o f G-6-P-dehydrogenase (Sigmaj 2 mg . / m l .) , and w ater to g iv e a f i n a l volume o f 2 .9 7 m l. were added to each c u v e tte . The r e a c tio n was s ta r te d by th e a d d itio n o f 0 .0 3 m l. o f th e s o lu tio n to be assayed and the red u ctio n o f TPN fo llo w ed in the sp e ctro ­ photometer a t 3U0 mp. The v a lu e s in th e ta b le are co rrected fo r th e i n i t i a l ab so rp tio n o f the complete system minus s u b s tr a te . S in ce t h i s enzyme d is p la y s a b so lu te s p e c i f i c i t y fo r G-6-P, th e con­ v e r s io n o f F-6-P to G-6-P by isom erase a c tio n was d e f i n i t e l y e s ta b lis h e d by th e h ig h er i n i t i a l r a te w ith the a liq u o t from the tube incubated fo r 20 m in u tes. U n fo rtu n a tely , th e presence o f isom erase a c t i v i t y in th e commercial p rep aration o f glu co se-6 -p h o sp h a te dehydrogenase i t s e l f made an accu rate estim a te o f the magnitude o f the conversion im p r a c tic a l. 55 I t should be n o ted , however,* th a t the r a te s o f red u ctio n were equal a f t e r 10 m in u tes. T herefore, a rough e stim a te was obtained by assuming th a t th e d if fe r e n c e in o p t ic a l d e n s ity between the two a ft e r 10 m inutes, when p lo tte d as 0 .D . v s . tim e, r e f le c t e d th e TPN r ed u ctio n due to th e in crea sed G-6-P in th e tube incubated w,ith glucosam ine sy n th e ta s e . This c a lc u la t io n was made in th e fo llo w in g manner (Horecker and Wood, 1957)'. 0 .0 5 1 x 3 ml* x 66 .7 ' -,/! ? 7 22 m ltia M --------- where 0 . 0 5 1 i s th e o p t ic a l d e n s ity d iffe r e n c e between the cu rv es, 6.22 ml./pM i s th e micromolar e x tin c tio n c o e f f ic i e n t o f TPN -at.310 .mji, 3 ml. i s th e volume in th e c u v e tte , and 66.? the d ilu t io n f a c t o r . The c a lc u ­ la te d v a lu e (1 .6 1 juM) fo r th e G-6-P which appeared was o f th e same order o f magnitude as the decrease in F-6-P (2 .1 5 pM) found by the anthrone method. The p resence o f the isom erase in the p u r ifie d glucosam ine synthe­ t a s e could th e r e fo r e e x p la in th e c a p a c ity o f 0 -6 -P to serve as a sub­ s t r a t e in th e r e a c tio n . This in t e r p r e ta tio n was strengthened by th e d e f i n i t e la g phase and reduced r a te obtained w ith G-6-P as compared to F-6-P in the tim e course stud y ( c . f . F ig . 6 ) . R eference to Table VIII provides a d d itio n a l support fo r th e fu n c tio n o f F-6-P as the primary su b s tr a te fo r th e B la s t o c la d ie lla s y n th e ta s e . The 50$ r ed u c tio n in th e G -6-P/F-6-P a c t i v i t y r a tio during p u r if i­ c a tio n , th e r e fo r e , e s ta b lis h e d w ith reasonab le c e r ta in ty th a t F-6-P was th e a c tu a l su b s tr a te fo r the B la s t o c la d ie lla glucosam ine sy n th e ta se , and th a t the react ion c a ta ly z ed by th e enzyme-was: 56 TABLE 71I I COMPARISON.OF THE EFFICIENCY OF GLUCOSE-6-PHOSPHATE AND FRUCTOSE-6-PHOSPHATE AS SUBSTRATES FOR/THE GLUCOSAMINE- SYNTHETASE .REACTION DURING „ PURIFICATION S p e c if ic A c t iv it y w ith G-6-P „ S p e c if ic A c t iv it y w ith F-6-P P u r if ic a t io n S tage Supernatant 93% Protamine supernatant . 81$ Gel e lu a te '(p h o sp h a te ) C on d ition s: h7% The enzyme f r a c t io n was incubated w ith 20 pM F -6-P , or G -6-P, 20 pM glutam ine, 100 pM phosphate b u ffe r , pH 6 .5 ; • T = 3 0 .6 C. F in a l volume 2 .0 ml - The r e a c tio n was stopped w ith 0 .0 5 '^1* I N HC1 and 0 .0 1 ml. 7% Na2W04 , and th e GA-6-P analyzed by the. method o f D isch e. . d -fr u c to se -6 -p h o sp h a te + 1 -g lu ta m in e >- d -glucosam ine-6-p hosphate + 1 - gl.utamate The pH optima and su b str a te requirem ents were the same as th o se reported fo r th e enzyme from Neurospora (L e lo ir and C ardini, 1953; Blumenthal., e t . a l . , 1955)* The data a ls o e sta b lis h e d th a t the B ia s t o c l a d ie lla enzyme was not th e same as th a t reported fo r ra t l i v e r by P o g e ll and Gryder (1957) j s in c e th e l a t t e r enzyme had a pH optimum between 7 -b and 8 .0 and dem onstrated an in c r e a s in g s p e c i f i c i t y toward G-6-P on p a r tia l p u r if ic a t io n . I t should be noted in c lo s in g th a t th e 1 9 -fo ld p u r if ic a tio n a tta in e d r e s u lte d in an enzyme preparation w ith a s p e c if i c a c t i v i t y (pM GA/mg. p r o te in /2 0 m in.) more than 2l;2 tim es th a t rep orted by L e lo ir and C ardini ( in pM GA/mg. p r o t e in / h r .) , and c a . If) t in e s th a t rep orted by B lum enthal, e t a l . , the o n ly oth er p u r if ic a tio n s pu blished to d a te . S tu d ie s o f R .S . Ontogeny. . The p u r if ic a t io n o f th e enzyme o u tlin e d in th e preceding s e c t io n , was undertaken w ith th e express purpose o f determ ining optim al condi­ t io n s fo r i t s a ssa y in p la n t hom ogenates. With th a t in form ation in hand, i t became p o s s ib le to proceed w ith a study o f the enzyme at d if f e r e n t s ta g e s in th e l i f e c y c le o f the fu n g u s. Glucosamine S yn th etase A c t iv it y in Z oospores, O.C. P la n ts , and R »S. P la n ts .— As a f i r s t approach i t was decided to compare the a c t i v i t y alrea d y determ ined fo r the mature th in -w a lle d p la n ts w ith th a t in th e mature R .S . p la n ts and in swarmers. To obtain a s u f f i c ie n t number o f swarmers f o r an enzymatic a ssa y , e ig h te e n 15 cm. P e tr i p la te s o f PYG agar were h e a v ily in o c u la te d w ith B la s t o c la d ie lla swarmers „ A fte r 12 hours -a dense su sp en sion o f spores was c o lle c te d from each p la te by a s e r ie s o f c e n tr ifu g a t io n s , u sin g the procedure d escrib ed by McCurdy (1959) • The swarmers were kept in an ic e -b a th a t a l l tim es from t h is sta g e on . c o ld 5 x 10 The c e l l s were f i n a l l y washed tw ice w ith 5 to 10 m l. o f _ 2 M phosphate b u ffe r , pH 6 .5 , by c e n tr ifu g a tio n a t 1600 x g . fo r 15 seco n d s. The packed c e l l s were resuspended in th e same b u ffe r (1 v o l . c e l l s : 9 v o l . b u f f e r ) , tr a n sfe r r e d to a sm a ll g la ss hom ogenizer, and ground u n t i l b e tte r than 95$ o f the c e l l s were broken. The homogenate was c en tr ifu g e d fo r 30 minutes at 22,000 x g . in a S e r v a ll Model SS-1 c e n tr ifu g e a t 2° C. The c le a r supernatant was then assayed f o r . i t s glucosam ine sy n th eta se a c t i v i t y . such experim ents are shown in Table IX. The r e s u lt s o f two 58 TABLE TX GLUCOSAMINE SYNTHETASE ACTIVITY IN ZOOSPORES S p e c if ic A c t iv it y With F- 6 -P With G-6-P Volume o f C e lls P ro tein C oncentration Exp. 67 0 .1 3 m l. 3 .82' mg ./m l. 2.2 1.6 Exp. 68 0 .2 6 m l. 3 -55 m g ./m l. U-3 3-2 C o n d itio n s: The supernatant (0 .2 m l.) was incubated w ith 20 pM glu ­ tam ine, 20 pM F - 6 -P , or G-6 -P , and 100 jaM phosphate b u ffe r , pH 6 .5 , in a f i n a l volume o f 2 .0 m l., fo r 20 min'.j T = 30 C. • The r e a c tio n was stopped w ith 0 .0 6 ml. 1 N HC1 and 0 .0 1 m l. 7 % Na2WQ4 ,- and th e GA-6 -P determined by th e D ische method. A ll v a lu es were co rrected fo r unincubated c o n t r o ls . 1 S p e c if ic A c tiv ity : uM GA-6 -P/m g. p r o te in /2 0 ■min. To a ssay th e sy n th eta se a c t i v i t y in mature R .S. p la n ts , two 1 .5 2 l i t e r c u ltu r e s c o n ta in in g 2 .3 8 x 10~ M sodium bicarbonate were in o cu la ted w ith swarmers and grown fo r 1 days a t room tem perature. The mats o f mature r e s is t a n t sporangia d eriv ed therefrom were homogenized in O 5 x 10~~M phosphate b u ffe r , pH 6 .5 in th e Omni-mixer. Follow ing c e n t r i­ fu g a tio n ( 22,0 00 x g . , 30 m in .), th e supernatants were analyzed fo r t h e ir sy n th eta se a c t i v i t y w ith th e standard, a ssa y procedure (Table X ). D ia ly s is of th e supernatant decreased th e a c t i v i t y c o n s id e r a b ly . In ad­ d i t i o n , th e sedim ent c o n s is t in g o f th e fragments o f th e r e s is t a n t sp oran gia and o th er c e ll u la r d eb ris was resuspended in b u ffe r and mixed in equal proportion s w ith th e su p ern atan t. This r e c o n s titu te d m ixture, c o n ta in in g the same q u a n tity o f supernatant as the u n r e c o n stitu te d m ixtu re, was a ls o a ssa y ed . ^ i t h e x cep tio n o f th e q u a n tity o f enzyme, t h is a ssay procedure was used fo r a l l subsequent d eterm in ation s o f glucosam ine sy n th eta se a c t i v i t y and w i l l be referred to as th e '■standard a ssay 59 TABLE X GLUCOSAMINE SYNTHETASE ACTIVITY OF MATURE R .S . P r o te in C oncentration S p e c ific A c tiv ity With F-6-P With G-6-P Exp. 69 Supernatant , 2 .0 mg ./m l. 0 .6 8 O.lU Exp. 70 Supernatant 2 .3 mg ./m l. 0 .6 2 0 .1 8 Exp. 70 R e co n stitu te d 0 .6 0 Exp. JO D ialyzed Supernatant3" 2 .0 mg ./m l. 0.1;5 cl — 2 .0 m l. supernatan t d ia ly z e d a g a in st 200 m l. 5 x 10 M phosphate b u ffe r , pH 6-5* fo r 7*5 hours at 2°C . The f a il u r e -of th e r e c o n s titu te d homogenate to dem onstrate in ­ c reased a c t i v i t y oyer th e supernatant a lo n e , e sta b lis h e d th e e x c lu siv e presence o f th e enzyme in th e so lu b le p ro tein s o f the su p ern atan t. This agreed w ith th e r e s u lt obtained e a r li e r fo r 'the O.C. p la n ts . The average s p e c i f i c a c t i v i t i e s fo r glucosam ine sy n th eta se in th e th re e p la n t forms are l i s t e d in Table 1 1 . TABLE XI A COMPARISON OF GLUCOSAMINE SYNTHETASE ACTIVITY IN ZOOSPORES, O.C. PLANTS, AND R .S . PLANTS Plant Tvt>e Plant Jype -W_________ ^ e q m ^ c t i v iWith t ^ _______ ith F_ 6_ p G-6-P Zoospores 3 *3 2 ,h T hin-w alled O.C. P lan ts 1*9 1*8 T h ick -w alled R .S . P lan ts 0 .7 0 .2 60 The d if fe r e n c e s in a c t i v i t y o f th e enzyme among the p la n t forms im m ediately r a ised th e q u estio n o f i t s r e la t io n s h ip to th e d iffe r e n c e s in th e form and development o f th e organism . problem;, T h is, th en , posed the did the d ecrea se in th e enzyme a c t i v i t y in mature p la n ts r e f l e c t i t s importance in the p ro cesses le a d in g to the g e n e sis o f a p a r tic u la r form o r , r a th e r , j u s t th e r e s id u a l a c t i v i t y a f t e r growth and s y n th e s is had ceased? To d ecid e which o f th e s e in te r p r e ta tio n s was c o r r e c t, i t was n e c e ssa r y to determ ine where the d ecrea se occurred during development and,, i f p o s s ib le , to c o r r e la te that knowledge w ith th e changes in oth er p ro cesses a s so c ia te d w ith growth. . Because o f a basic, in t e r e s t in th e g e n e sis o f th e r e s is ta n t 'sporangium, and th e in crea sed c h it i n s y n th e s is in v o lv ed in i t s development ( C antino, L o v ett, and H o ren stein , 1 9 5 7 ), th e s tu d ie s which fo llo w are concerned w ith t h is form o n ly . C u ltu ral C onditions fo r Synchronized Growth. — In order to study th e r e la t iv e a c t i v i t y o f th e .sy n th eta se enzyme a t d if fe r e n t sta g e s du rin g th e development o f R .S . p la n ts , two experim ental c o n d itio n s had to be s a t i s f i e d : (1) The growth o f the p lan ts had to be w e ll enough synchronized so th a t m ost, i f not a l l , o f the p la n ts were at th e same s ta g e o f growth a t any given tim e; (2) C ultures had to be grown in a manner which perm itted removal o f a r e p r e se n ta tiv e sample a t d e sire d in te r v a ls . The u se o f swarmer su sp en sion s p a r t ia lly s a t i s f i e d th e f i r s t re­ quirement . It has r e c e n tly been shown th a t such su sp en sion s when placed in liq u id media germ inate in e s s e n t i a ll y synchronous fa sh io n 61 ( Turian and C antino, 1 9 5 0 ). In an attem pt to s a t i s f y th e second con- d it io n a s e r ie s o f id e n t ic a l 3 ~ H te r fla s k s (c o n ta in in g 2 .3& x 10 - 2 M NaHC03 b efo re a u to c la v in g ) were in o cu la ted w ith a uniform number o f m o tile swarm ers. Each f la s k was h a rv ested a t a d if f e r e n t tim e . However, a f t e r s e v e r a l runs were made, u s in g the c o n d itio n s d escrib ed above, r e s u lt s in d ic a te d th a t n e ith e r o f the two c r i t e r i a was in f a c t b ein g s a t i s f a c t o r i l y m et. The f i r s t and more se r io u s problem involved th e -2 c o n d itio n o f the p la n ts grown in 2.38 x 10 " M sodium b ica rb o n a te. The c o n c e n tr a tio n used was o r ig in a lly chosen because i t ensured 100$ R .S . form ation on agar c u ltu r e s . But in liq u id c u ltu r e s , although some p la n ts developed in an ap p aren tly normal fa s h io n , oth ers appeared normal a t f i r s t but showed in c r e a sin g abnorm ality and d egen eration a fter , approxim ately L|h hours o f growth. M orp hologically, th e most obvious changes were a pronounced abnormal th ick en in g o f th e c h itin o u s sp o r a n g ia l w a ll (F ig . 8 ), depressed pigm en tation , and u lt im a te ly , a clumping o f th e sp o r a n g ia l c o n te n t s . T herefore, a s e r ie s o f f la s k s were s e t up to e s t a b lis h th e optim al co n d itio n s fo r a proper balance between R .S , form ation and h e a lth y growth in liq u id media. As a consequence, _3 i t was found th a t 8 .9 x 10 M sodium bicarbonate autoclaved in th e PYG medium y ie ld e d th e b e st r e s u lt s ; c u ltu r e s grown under th e se c o n d itio n s c o n s is te d o f b e tte r than 98 $ R .S. p la n ts and th ese appeared e n t ir e ly normal throughout t h e ir developm ent. The second problem encountered w ith th e s e r i a l f la s k tech n iq u e was u n s a t is f a c to r y r e p r o d u c ib ilit y in ,grow th r a te s among r e p lic a t e f la s k s , although a l l th e c o n d itio n s were made as n e a r ly id e n tic a l, as p o s s ib le , 62 Figure 8 A Thick-W alled R .S. P lan t Produced in Super-Optimal B icarbonate C oncentrations 63 in c lu d in g th e s i z e o f inoculum , tem perature, and t h e .r a t e o f a e r a tio n . The degree o f clumping o f p la n ts had a n o tic e a b le e f f e c t on growth. S e v e r a l en tan gled p la n ts always matured more r a p id ly than s in g le on es, and t h is e f f e c t could be d e te c ted f o r even a s in g le p a ir o f p la n ts . The- v a r i a b i l i t y in degree o f clumping could not be elim in a ted w ith ou t red ucing th e s i z e o f ihe inoculum to an im p ra ctica l l e v e l . To o b v ia te t h i s d i f f i c u l t y fla s k s were used o f s u f f i c i e n t s iz e (12 l i t e r s ) to accommodate enough medium (10 l i t e r s ) fo r sam pling throughout th e e n tir e growth p e r io d . The r e s u lt s obtained by t h is tech n iq u e were e x c e lle n t . In such c u lt u r e s , the great m a jo rity o f p la n ts grew s in g ly or at most in tw o's and t h r e e 's 'throughout, th e generation, tim e . Growth S tu d ie s w ith Synchronized C u ltu r es-.— To e s t a b lis h th a t th ese c u ltu r e s were as w e ll synchronized as th ey appeared to be by v is u a l ex­ am in ation, samples were removed fo r s iz e measurement approxim ately every 6 hours up to 60 h ou rs, and a t l e s s frequent in t e r v a ls t h e r e a f t e r . The appearance o f p la n ts a t th e se d if f e r e n t sta g e s o f development is shown in Figure 9 • Twenty p la n ts from each sample were measured at random... At th e e a r lie r sta g e s when th ey were sp h e r ic a l or e l l i p s o i d a l in sh ap e, on ly th e le n g th and w idth was recorded. For th e o ld e r d evelop ­ m ental sta g e s a f t e r th e i n i t i a t i o n o f th e sporangium, th e o v e r a ll le n g th and the diam eter o f both sporangium and s t a lk were recorded (T able X I I ). C a lc u la tio n o f th e average volume per p la n t at each sta g e during growth was based upon th e assum ption th a t the shape o f th e p la n ts was s u f f i c i e n t l y near to th a t o f a sphere so th a t a s im p lifie d c a lc u la tio n would not cause s e r io u s e r r o r . The volume o f p la n ts a t d if f e r e n t ages Figure 9 Photomicrographs o f S .S . P la n ts During Development in Synchronous Culture ( X 350 ) 65 66 67 TABLE XII SIZE MEASUREMENTS OF DEVELOPING R..S. Age in Hours 0 (Z oospores) 8 1 2 .5 18 • ' 21+ Length (p) Diameter o f Sporangium (jj) 9 Diameter o f S ta lk (ji) 7 ■ 1 6.5 16.1+ 2 9 .5 29.2 5 i . 1+ 51.1 92.5 ' 92.0 ■ 3 0 .5 193.7 11+2.6 36 210 .2 151 *1 111+.8 k2 212 .1+ , 152.7 111+.8 1+8 210 .7 11+6-6 1 0 2 .5 51+ 207.6 11+5 *li 101.8 60 ' 209.5 • 1 I+6 . 3 . 103 .1 72 209.5 1 3 9 .6 1 0 0 .6 107 202.7 11+2.5 9 7 .2 . • Each v a lu e rep re sen ts th e average fo r 20 p la n ts measured a t random. The v a lu e s •within- any one sample were s u f f i c i e n t l y c o n s is t e n t to make a s t a t i s t i c a l a n a ly s is un necessary. i s p lo tte d in Figure 10A-B. The ob serv a tio n s th a t th e p lo t o f the r e la t io n s h ip between volume and age was sigm oid , and th a t th e lo g a rith m ic p lo t y ie ld e d a s tr a ig h t l i n e were taken as a d d itio n a l confirm ation o f synchronized growth. I t i s im portant to note th a t the in c r e a se in s i z e f a l l s o f f r a p id ly a f t e r 30 hours and a d ecrease in s iz e even seems to occur; the l a t t e r must be due to a c e r ta in amount o f shrinkage during th e m aturation p r o c e ss, but i t s cause remains unknown. To o b ta in one fu r th e r parameter concerning sy n ch ro n iza tio n o f th ese c u lt u r e s , th e dry w eight p e r t h a ll u s was determined as a fu n c tio n of 68 p la n t a g e . It was im p o ssib le to use .1 2 -lite r f la s k s fo r th e s e determ in­ a tio n s because a co n sid era b le q u a n tity o f plant m a ter ia l adhered to th e upper p o rtio n s o f the f la s k and made q u a n tita tiv e r e c o v e r ie s by th e siphon procedure u n r e lia b le . For t h is reason a s e r ie s o f 3 ~ l i t e r fla s k s were in o c u la te d w ith a known q u a n tity ( i . e . , few enough to prevent over­ crowding) o f swarmers and grown under id e n t ic a l c o n d itio n s . The r e s u lt s are given in Table X III. The dry w eigh ts of p la n ts a t d if f e r e n t ages were obtained from th e d a ta in Table X III (F ig . 11A-B) . The sigm oid and s tr a ig h t l i n e r e la t io n s h ip s between dry w e ig h t/p la n t and tim e, and lo g dry w e ig h t/ p lan t and tim e , r e s p e c t iv e ly , were c o n s is te n t w ith a s a t is f a c t o r y sy n ch ro n iza tio n during the growth'and development o f th e R .S. p la n ts . For purposes o f comparison which w i l l become ev id en t in th e d is ­ c u s s io n , an arrow has been placed on subsequent graphs in d ic a tin g th e age (3 6 h r .) where th e in c r e a se in s i z e o f p lan ts cea sed . Glucosamine S yn th etase A c tiv ity During R .S . Developm ent.—With con­ d it io n s e s ta b lis h e d fo r growing synchronized, rep ro d u cib le, mass c u ltu r e s o f R .S . p la n ts , th e stud y o f glucosam ine sy n th eta se a c t i v i t y during development was co n tin u ed . The d a ta from a ty p ic a l experim ent u sin g such c u ltu r e s are d e lin e a te d in Figures 12, 13, and lljA-B. The c u ltu r e f l a s k , incubated at 2 k ° C ., was sampled by removing approxim ately one and o n e -h a lf l i t e r s o f th orou ghly suspended plant m a teria l a t f i v e in t e r v a ls between 2 1 .5 .and 83 hou rs. In order to o b ta in enough p lan t m a te r ia l fo r th e 12 hour d a ta , i t was n ecessa ry to use a [ [ - l i t e r f la s k 69 TABLE X III DRY WEIGHTS OF PLANTS AT DIFFERENT AGES DURING R .S. DEVELOPMENT T o ta l Number o f P lan ts T otal Dry Weight ( grams) 5 3 2 ,972,510 0.0602 251+, 1 0 7 ,81+0 1.0297 21+ 3,1+93,983 0 . 31+2.2 36 1 ,2 3 3 ,1 7 0 O . 773 O 1+8 1 ,2 3 3 ,1 7 0 1.061+9 60 1 ,2 3 3 ,1 7 0 1.0311+ 81+ ■■ ■ 1,1538,699 1.170-7 Age in ..Hours 0 (Z oospores) 12 ’ C on d ition s: The swarmers were h arvested as p r e v io u sly d escrib ed fo r enzyme p rep a ra tio n s, washed w ith d i s t i l l e d w ater, and d ried to c o n sta n t w e ig h t. The 12 h r . p la n ts were grown in a l+ ~ lite r 'fla s k w ith PYG plu s 8 .9 x 10 “ 3 M NaHC03 , th e r eg a in in g c u ltu r e s were grown in 3 ~ l it e r f la s k s o f th e same medium] a l l a t 23 ± 1° C. w ith a much h e a v ie r inoculum than could be used f o r 't h e la r g e f l a s k s . From each, a liq u o t o f h arvested p la n ts a sample was taken fo r a dry weight' d eterm in a tio n . The remainder was homogenized in th e Omni-mixer —2 w ith 5 -x 10 M phosphate b u ffe r , pH 6 .5 , and the homogenate cen trifu g ed f o r 30 minutes a t 22,000 x g . The supernatant was separated from th e sedim ent and an upper la y e r o f l i p i d i c m a teria l w ith a p i p e t t e . Each supernatant was th en assayed fo r i t s glucosamine sy n th eta se a c t i v i t y w ith the standard assay procedure, u sin g approxim ately 0 .6 mg. o f supernatan t p r o te in per tu b e . The averaged d ata from s e v e r a l s e r ie s o f c u ltu r e s grown in s in g le 3 - l i t e r f la s k s co n ta in in g the h igh er co n cen tra tio n s o f bicarbonate 70 F igure 10A-B. The Volume-of an R .S . Plant During Development. . P lan ts were assumed to be sp h e r es, and '’average" diam eters were d erived from th e d ata in Table X II, F ig . 10A; Volume per P la n t. F ig . 10B; Log^-Volume per P la n t. F igure 11A-B. The Dry W eight o f an R .S. P lant During Development. Data were c a lc u la te d as th e t o t a l dry w eight per c u ltu r e / th e number o f p la n ts per c u ltu r e . F ig . 11AJ .Dry Weight per P la n t. F ig . llB j Log-Dry Weight per P la n t. F igure 1 2 . The S p e c if ic A c t iv it y o f Glucosamine S y n th eta se in B..S. P lan ts During Developm ent. S p e c if ic A c tiv ity : pM GA-6 -P/mg. p r o te in /2 0 min. co n tex t fo r d e t a i l s ) . (See Figure 1 3 . T o ta l Glucosamine S y n th eta se per U nit Weight o f Organism During R .S . Development. One u n it o f sy n th eta se : th e q u a n tity o f enzyme m ediating th e production o f •1 pM GA-6-P/20 minutes in th e standard a ssa y ( s e e c o n te x t). T otal u n its sy n th eta se per gm. dry w eight: t o t a l g m ..so lu b le p ro tein x u n its per gmi p r o te in / gm . dry w eight'. F igure II4A.-B . Glucosamine S yn th etase A c tiv ity per P lan t During R .S. D evelopm ent. F ig . liiAj S yn th etase U nits per P lant ( t o t a l u n its per gm. dry w eight (F ig . 13) x gm. dry w eight per plant (F ig . 11A ). F ig . lUBj L og-Synthetase U nits per P la n t. 71 -2 268 F igu re 10A X 192 144 96 _J -6 48 24 -7 72 96 24 AGE, HRS. AGE, HRS. to o Figure 1133 Figure 11A •o 60 +2 60 >- +1 > -40 20 Oo. 72 24 A G E, HRS. 48 AGE, HRS. 72 F igure 12 750 5.0 O CL 2 . 0 1.0 F-6-P 1—200 o F-6-P 0- 5 100 G-6-P G-6-P 40 o 0 72 72 24 AGE, HRS. Figure 14 b F-6-P 2 100 F -6 - R, G-6-P 50 O G-6-P i 72 24 AGE, HRS. 24 48 72 *38 x 10 M) in th e medium, but harvested and analyzed in an id e n t ic a l manner to the la r g e c u ltu r e s , have been p lo tte d in Figure 15> fo r com­ p a r iso n . The p a ttern o f the curves i s e s s e n t ia ll y the same in both c a ses; how ever, i t i s im portant to n o tic e th at the peaks in th e curves are d elayed alm ost e x a c tly 21; hours by the 2 . 7- f o ld in c r ea se in the b icarb on ate c o n c e n tr a tio n . By m icroscopic o b serv a tio n i t was esta b ­ lis h e d th a t the m orphological changes a sso c ia te d w ith R .S. development were a ls o s h if t e d by th e same amount. The s ig n if ic a n c e o f th e peaks in t o t a l and s p e c i f ic a c t i v i t y and t h e ir .d if f e r e n t ia l appearance w ith r e sp e c t to th e age o f th e p la n ts w i l l be d isc u sse d .la te r . However, i t i s important to poin t out now th a t th e ste a d y d ecrea se in a c t i v i t y found w ith G-6-P as the su b str a te fo r th e sy n th e ta se enzyme, 'as compared to F- 6 -P , was q u ite s t r ik in g . From th e r e l a t i v e a c t i v i t i e s o f the two su b str a te s during the p u r if i­ c a tio n procedure, and from th e r a te s t u d ie s , i t had been concluded th a t th e G-6 -P was converted to F - 6 -P by phosphoglucose isom erase a c tio n , r a th e r than i t s e l f d ir e c t ly involved in th e r e a c tio n . I f , in d eed , t h is w e re -th e case the stead y decrease in a c t iv i t y could be in ter p r e te d as r e f l e c t i n g a d ecrease in th e a c t i v i t y o f th e isom erase during d if fe r e n ­ t i a t i o n , thus gra d u a lly b lo ck in g th e in ter c o n v e rsio n between th e two hexose-p h osp h ates . To t e s t t h is h y p o th esis i t was n ecessa ry to e sta b ­ l i s h whether th e phosphoglucose isom erase did decrease in a c t i v i t y during grow th. The sim p le st method fo r doing th is was to measure th e red u ctio n o f TPN by g lu c o s e - 6- phosphate dehydrogenase in B la s t o c la d ie lla e x t r a c t s , u s in g each hexose-phosphate as s u b s tr a te . The r e la t iv e a c t i v i t y o f the two in such a system would in d ic a te th e a c t i v it y o f th e isom erase s in c e i t s p resence would be required fo r th e con version o f F-6-P to G -6-P. The glu co se-6 -p h o sp h a te dehydrogenase in turn had an a b so lu te s p e c i f i c i t y fo r G-6-P and TPN. The method would on ly be v a lid i f th e dehydrogenase were p r e se n t, and G-6-P thus served not on ly as a reference, fo r the r a te s of red u ctio n w ith F -6-P , but a ls o as a c o n tr o l fo r th e presence o f th e dehydrogenase i t s e l f . G lu co se-6 - phosphate Dehydrogenase and. Isomerase A c tiv ity During R .S . Development ■— This experiment was done w ith swarmers, a 12 hour R .S . c u ltu r e , and a la r g e synchronized R .S. c u ltu r e , a l l grown and h a rv ested as p r e v io u sly d e sc r ib e d . With th e r e s is t a n t sp oran gia! p lan ts a sample o f the mat was used fo r a dry w eight d eterm ination and the remainder homogenized in 5 x 10 M phosphate b u ffe r , pH 7*0* The swarmers were h arvested and homogenized as u su al in th e same b u ffe r . One p o r tio n o f th e whole homogenate was cen trifu g ed fo r 30 minutes at 22,0 0 0 x g . and used fo r p r o te in and n itro g e n determ inations (s e e la t e r ) A second 3 m l. p o rtio n was d ia ly z e d a g a in st 2 l i t e r s o f the same b u ffe r fo r 10 hours at 2° C. A fter d i a l y s is th e m a teria l was q u a n tita tiv e ly recovered and d ilu te d to a volume o f 10 ml. w ith b u ffe r . The d ilu te d homogenate was cen tr ifu g e d at 500 x g . fo r 10 minutes and the p r o te in co n c en tr a tio n o f the supernatant determ ined. The r a te o f the g lu c o se- 6 - phosphate dehydrogenase r e a c tio n was measured by fo llo w in g th e red u ctio n o f TPN at 3bO mp (Table XIV) . The q u an tity o f p r o te in added to each c u v e tte {.ca. O.Oii. to 0 .1 mg.) was adjusted to y ie ld r a te s of red u c tio n that could be determ ined a c c u r a te ly , and which remained lin e a r f o r at l e a s t 10 to 15 minutes . The changes in the o p t ic a l d e n s ity \_0.D.) per 10 m inutes ranged from 0.21+ to 0.81+ in th e a ssa y system u se d . No TPN r ed u ctio n was observed in co n tr o ls w ithout added sub­ s t r a t e , in d ic a tin g th a t the d ia l y s i s had removed endogenous s u b str a te s . - TABLE XIV GLUCOSE -6-PHOSPHATE DEHYDROGENASE ACTIVITY DURING DEVELOPMENT OF R.S'. PLANT'S Age in Hours ' Mg. P ro tein Used in Assay Rate o f TPN R eduction as A 0 .D ./m in .a With 0-6-P With F-6-P 0.01+5 0 . 051+ 0.028 12 0.102 0.020 0.013 21+ ■ 0.029 0.021+ '36 0.055 0 .0 5 6 0 . 0 I+5 0 .0 3 1 1+8 0.01+8 0.01+1+ 0.031+ 60 0 .0 5 1 0.01+9 . 0.036 83 0 .0 3 7 0 .0 7 0 0.01+6 0 (Z oospores) C ond itions: Each c u v e tte contained 0 .6 m l. 10“ ^-M phosphate b u ffe r , pH 7 .5 , 0 .1 m l. 10“ XM MgCl2, 0 .2 ml. 2 -5x 10“3M TPN, 0 .3 ml. 10“ ^-M F -6-P , or G-6-P, 0 .0 5 to 0..Q8 ml. enzyme, and water to a f i n a l volume o f 3 .0 ml. The r e a c tio n was s ta r te d by the a d d itio n o f th e enzyme, and the O.D. read each minute, beginning at 2 m in ., in a Beckman Model DU spectrophotom eter at 3 I4O mp. The blank cu v e tte lacked su b strate, and TPN. ®The r a te was c a lc u la te d fo r the 3 -6 min. in t e r v a l (from p lo ts o f O.D. v s . tim e) because the r a te s o f red u ctio n w ith F -6-P, which d isp la y ed an i n i t i a l la g , became lin e a r and maximal a f t e r 2 min. The s p e c i f i c ' a c t i v i t i e s obtained fo r the p la n ts at each age are p lo tte d in Figure 1 6 , and an ex p ressio n o f the t o t a l u n its o f dehydro­ genase a c t i v i t y per plant as a fu n c tio n o f age in Figure lyA -B . 76 From an exam ination o f th e curves i t appeared that F-6-P did gradually l o s e some o f i t s c a p a c ity to fu n c tio n in th e coupled system . To t h is e x te n t, i t seemed to confirm th e su ggested lo s s in isom erase a c t i v i t y du rin g development . However, i t was evident from th e comparative data in Table XIV th a t th e d ecrease in isom erase a c t iv it y could not adequ ately e x p la in a l l th e change in the c a p a c ity o f G-6-P to serv e as a su b str a te in th e sy n th e ta se r e a c tio n . TABLE XV THE. RELATIVE ACTIVITIES OF FRUCTOSE-6 - PHOSPHATE AND GLUC0SE-6-PH0SPHATE IN THE GLUCOSAMINE SYNTHETASE AND GLUC0SE-6-PH0SPHATE DEHYDROGENASE ENZYME SYSTEMS DURING R .S . DEVELOPMENT Age o f P lan ts (h o u rs) (1 ) R atio 'P~L" ~p A c tiv ity in S yn th etase R eaction (2 ) R atio y £ -p A c tiv ity . in Dehydrogenase ^.2)-^l) R eaction 0-73 0 .5 2 -0 .21 12 0 .3 7 . 0 .6 6 0.09 2h 36 0.7U 0 .81+ 0 .1 0 0.63 0 .8 2 0 .19 U8 0.33 0 .7 7 0.21+ 60 0.33 0.7U 0.1+1 83 0 .17 0 .6 7 0 .3 0 0 (Z oospores) The r a t io s were the same fo r both s p e c if ic and t o t a l a c t i v i t y . I f th e isom erase were th e o n ly lim itin g fa c to r in th e p a r tic ip a tio n o f g -6 -P in th e sy n th e ta se r e a c tio n the r a tio s in columas (1) and { 2 ) should have been, o f th e same order o f magnitude. im m ediately obvious th a t t h is was not the c a s e . However, i t was A p o s s ib le in te r p r e ­ t a t io n o f th e anomalous behavior o f G-6-P w i l l be d isc u sse d l a t e r . 77 F ig u re 15* T otal Glucosamine S yn th etase per Unit Weight o f Organism During F.. D. Development in Super-optim al B icarbonate Concent rat i o n s . B icarbonate co n cen tra tio n : fo r c a lc u la t io n s . Figure 1 6 . 2.38 x 10 _ 2 M. See Figure 13 The S p e c if ic A c tiv ity o f G lu cose-6 - phosphate Dehydrogenase in R .S . P lan ts During Developm ent. S p e c if ic A ctivity .; r a te o f TPN red uction/m g. p ro tein (s e e a ssa y procedure Table XIV). Figure 17A-B. G lu cose-6 - phosphate Dehydrogenase A c tiv ity per P lant During R .S . D evelopm ent. One u n it o f dehydrogenase a c t iv it y : th e q u a n tity o f enzyme m ediating a A O.D. o f O .l/m in u te (s e e a ssa y procedure Table X IV ). The u n its per plant were c a lc u la te d by the procedure-described in Figures 13 and lU* Figure 1JA', Dehydrogenase U n its,p e r P la n t. Figure 17Bj Log-Dehydrogenase U nits per P la n t . Figure 1 8 . The C h itin Content per Unit Weight o f Organism During R .S. D evelopm ent. (See M aterials and Methods fo r d e t a i l s ) Figure 1 9 . The C h itin Content per Plant During R.S. Development. jxg c h it in -per mg. dry weight (F ig . 18) x mg. dry w eight per plan t (F ig . 11A ). Figure 2 0 . The Melanin Content per Unit Weight o f Organism During R .S. D evelopm ent. One u n it o f melanin:- th e q u a n tity o f m elanin y ie ld in g an O.D. o f 0 .0 0 1 a t I4.5O mp (see c o n te x t). Figure 21 . The Melanin Content per Plant During R.S. Development. U nits melanin per gm. dry weight (F ig . 20) x gm. dry w eight per plan t (F ig . U A ) . 78 2 .0 F igure 15 Figure 16 730 G-6-P u. 1.0 200 F-6-P Q 0.5 F-6-P o G-6-P 0 96 48 72 24 144 AGE, AGE, H R S HRS. o 150 F igure 1?A G-6-P G-6-P F-6-P +2 F-6-P UJ O+i Ll I UJ 72 24 AG E, HR S. 24 72 A G E , HR S . 79 1000 Figure 19 o 100 o X LOQ < a. 500 = 50 oa. o o -2 0 72 24 72 24 AGE, HRS. AGE, HRS. 60 100 X 10 Pigure 20 UNITS / PLANT to 6 0 MELANIN 60 LOG LlJ z o 60 AG E, HRS 12 36 60 AG E , HRS. 64 80 C h itin Formatio n In D eveloping R .S .— Among oth er th in g s , the study o f glucosam ine sy n th e ta se had been undertaken to d e te c t any c o r r e la tio n s between i t s a c t i v i t y and the fr e e glutam ic a cid p o o ls, and in d ir e c t ly th ereb y , th e s i t e o f bicarbonate f i x a t i o n in the c i t r i c a cid c y c le . With the sy n th eta se inform ation a v a ila b le i t became d e s ir a b le to examine th e other end o f t h is b io s y n th e tic pathway, namely th e f i n a l product o f th e sequence, c h i t i n . two th in g s : This could have accom plished ( 1 ) i t might have brought out any c o r r e la tio n between th e r a te o f s y n th e s is o f c h it in and the a c t iv it y o f th e glucosamine s y n th e ta s e , and (2 ) i t might have e sta b lis h e d the pattern o f c h it in s y n th e s is during d if f e r e n t ia t io n . The c h it in a n alyses at se v e r a l sta g e s during the growth period are shown in Figure 18 . I t i s worth emphasizing th a t the b im o d a lity of th e curve (F ig . 18) com p letely disappeared when th e d a ta were converted to a p er-p la n t b a s is (F ig . 1 9 ) t Previous mention was made o f th e fa c t th a t p la n ts grown in e x c e s s iv e ly high bicarbonate co n cen tra tio n s developed abnormally th ic k sp o ra n g ia l w a lls ( c . f . p . 6 1 ). To v e r if y th e se o b serv a tio n s q u a n tit a tiv e ly , m a teria l from such c u ltu re s was analyzed fo r c h it i n lia b le XVI). The r e s u lt s rev ea led th at the h igh er b icarb on ate c o n c en tr a tio n caused a 73 to 87$ in c r ea se over th e normal c h it i n content o f th e p la n t s . The c h a r a c t e r is t ic production o f the th ick ,, c h itin o u s w a ll in R .S. p la n ts was always accompanied by th e d e p o sitio n o f melanin in th is s tr u c tu r e , and by in creased fa t sy n th e sis . Because o f th ese r e la t io n - s h ip s , a study o f m elanin and l i p i d production during R .S. form ation was undertaken. 81 TABLE XVI THE CHITIN CONTENT OF R .S. PLANTS GROWN IN MEDIA WITH DIFFERENT SODIUM BICARBONATE CONCENTRATIONS Age o f P lan ts NaHC03 C oncentration 5 days 8 .93 x 10“ 3 M. 9 2 .5 7 days 2 .38 x 10" 2 M. 160.3' Normal, and th ic k w alled 5 days 2 .38 x 10"' M. 172.9 Abnormal"arid very th ic k -w a lle d pg C h itin per mg. Dry Weight Appearance o f P la n ts Normal M elanogenesis During R .S . Ontogeny.— It had alread y been e sta b ­ lis h e d (Cantino and H oren stein , 19£5c) th a t th e colored m a teria l in th e c e l l w a lls o f B la s t o c la d ie lla R .S. e x h ib ited the p ro p e r tie s ch aracter­ i s t i c o f m elanoid pigm en ts. T herefore, the course o f m elanogenesis during th e growth o f th e R .S . p la n ts was e sta b lish e d (F ig s . 20 and 2 1 ). E ig h t y - s ix p ercent o f the melanin sy n th esized was produced a ft e r growth in s i z e ceased ( i . e . , a f t e r 36 h r .) but the f i n a l y ie ld o f melanin was reached a t the same age (60 h r .) as was that fo r c h it i n . L ib id S y n th e sis During R .S . Development .— The in c r e a se in the q u a n tity o f l i p i d during growth was very obvious in the l i v i n g t h a l l i m ic r o s c o p ic a lly , and in thp homogenates of th ese p lan ts as a su rface la y e r a f t e r c e n tr ifu g a tio n . In mature R .S. th e l ip id s appeared to c o a le sc e in to la r g e g lo b u les ( c . f . F ig . 9 , 83 h r . ) . The a n a ly ses fo r t o t a l l i p i d s -vF ig s . 22 and 2 3 ) do not in clu d e data fo r th e spore sta g e 82 because i t was im p ra c tic a l to c o lle c t enough m a teria l fo r an accurate d e te r m in a tio n . As w ith c h it in and m elanin, a co n sid era b le proportion o f th e t o t a l l i p i d was sy n th esiz ed a f t e r the c e s s a tio n o f growth in s iz e . N itrogen Transform ation in D eveloping R .S. P la n ts .—S ig n ific a n t d if fe r e n c e s had been found between th e so lu b le amino acid pools o f O.C. and R .S . p la n ts ( c . f . p . 3U) • A previous rep ort (O antino, L o v ett, and H o ren stein , 1957) had a ls o in d ica ted a co n sid era b le d iffe r e n c e in other n itr o g e n -c o n ta in in g fr a c tio n s between the two typ es o f p la n t . For t h is reason th e d is t r ib u t io n o f n itro g e n pools was re-ev a lu a ted at. d if f e r e n t 1 s ta g e s during th e development o f R .S . p lan ts ( F ig s . 25, 26, and 2 7 ). I t was obvious th a t s ig n if ic a n t changes were occu rring in th e d i s ­ t r ib u t io n o f n itr o g e n during growth, and t h is was nowhere more apparent than in th e s o lu b le n o n -p ro tein n itr o g e n . At 36 hours t h is fr a c tio n a c t u a lly appeared to exceed the so lu b le p r o te in n itr o g e n , although i t r a p id ly decreased a g a in . . The f a c t th a t th is n o n -p ro tein fr a c tio n should have c o n s is te d p rim a rily of amino a cid s made a chromatographic survey o f the fr e e pools of these'compounds im p era tiv e. The r e s u lt s ( F ig . 28) revealed a s tr ik in g c o r r e la tio n between th e l e v e l s o f s o lu b le , n o n -p ro tein n itr o g e n and the q u a n titie s o f e x tr a c ta b le amino a cid s at d if f e r e n t ages in ontogeny. I t should be noted th a t in a d d itio n to the ^By a n a ly zin g supernatants b efo re and a ft e r TCA p r e c ip ita tio n i t was a ls o p o s s ib le to check the accuracy of the TCA-turbidometric method used to e stim a te p r o te in s . The a n a ly s is o f d ia ly ze d and un dialyzed ■ supernatan ts served to v e r if y the data obtained by the TCA method and thus v e r if y the s o lu b le p ro tein e s tim a tio n s . The correspondence o f the curves fo r p r o te in n itro g e n by a n a ly s is and by c a lc u la tio n from the TCA p r o te in d eterm in ation s i.Fi-g* 2 6 ) , showed the l a t t e r to be a s a t is f a c t o r y method w ith B ia s t o c la d ie lla m a t e r ia l. 83 F ig u r e 2 2. The L ipid Content per Unit Weight o f Organism During R .S. D evelopm ent. Figure 2 3 . The L ipid Content per Plant During R .S. Development. Micrograms lip id /m g', dry w eight (F ig . 22) x mg. dry w e ig h t/ p lan t ( F ig . 11A ). Figure 21*. R e la tiv e Enzymatic A c t i v it i e s per U nit P ro tein N itrogen During P. .S . D evelopm ent. R e la tiv e a c t iv it y : t o t a l u n its o f a c t i v i t y / p i ant *7 t o t a l pg s o lu b le p r o te in n it r o g e n /p la n t. Figure 25- Log P lo ts fo r D is tr ib u tio n o f N itrogen per P lant During R. .S . D evelopm ent. See F i g . 2 7 . Figure 2 6 . The D is tr ib u tio n o f N itrogen per Unit Weight o f Organism During R .S. Development. The t o t a l so lu b le n itro g e n (TSN) was determined on super­ natants. (s e e c o n t e x t ) . Two methods were used to estim a te th e s o lu b le p r o te in n itro g en (SPN) and so lu b le n o n -p ro tein n itr o g e n (SNPN): (1) the supernatants were trea ted w ith 2 .%% TCA, the TCA-soluble n itro g e n determined (SNPN), and th e SPN obtained by d iffe r e n c e (TSN-SNPN)j (2) the super­ n a ta n ts were d ia ly ze d e x h a u s tiv e ly , and n o n -d ia ly za b le p r o te in n itro g e n determined (SPN), and the SNPN obtained by d iffe r e n c e (TSN-SPN). The p g n itro g en per mg. dry weight was obtained by d iv id in g the t o t a l q u a n tity in each fr a c tio n by the mg. dry w eight used fo r hom ogenization. The data obtained by the two'methods (two sep arate experim ents) were averaged fo r th e curves in th e f ig u r e . The c a lc u la te d p r o te in n itro g e n was obtained by assuming a n itro g en content o f 16$ fo r th e p ro tein s estim ated by the TCA-turbidometric m ethod. Figure 2 7• The D is tr ib u tio n o f N itrogen per Plant During R.. S.' Development .• The pg TN, SPN, or SNPN per plant were obtained from: p g TN, SPN, or SNPN/mg. dry w eight ;F ig . 26) x mg. dry w e ig h t/ plan t (F ig . 11A ). 200 F igure 22 Figure 23 LOG 190 / PLANT X 10 200 LOG jiG LIPID Q jO O 100 50 12 36 12 84 60 AGE, HRS. 20 60 AGE, HRS. 3 TN Figure (H SPN o ISOMERASE o Figure 25 -2 72 24 AGE, HRS. A G E , H rV 84 85 VO •H IM 0 A d a *9 IN / N 3 9 0 d ± I N 9 * m 4 l±J -01 oO 01 X l N V I d / N 3 9 0 d l l N 9* 86 Figure 2 8 . The S o lu b le Amino Acids of R. S. P la n ts During Development. The zoospore chromatogram was prepared w ith an ex tra ct o f c a . 3 ,U2 mg. dry w eight o f spores; the 12 to 83 hour p lan t chromatograms, an ex tra ct o f 3 mg. dry weight . Chroma­ tography was ca rried out in the lo n g d ir e c t io n w ith phenol■w ater, in th e sh o rt d ir e c t io n w ith b u tan ol-p rop ion ic a c id w ater, and th e amino acid s d e te c ted w ith n in h yd rin . 83 gen eral changes in th e t o t a l amino acid l e v e l s , c e r ta in in d iv id u a l compounds in creased or decreased d i f f e r e n t i a l l y . fo llo w in g should be mentioned: In p a r tic u la r , the (1) the r e l a t i v e l y high pools o f both a la n in e and glutam ate throughoutj (2) the appearance of asparagine at 83 hours; and ( 3 ) th e sharp r is e in the glutam ate pool at 83 hou rs. In s h o r t, a co n sid e ra b le r eo r g a n iz a tio n of the pathways le a d in g to and from th e s o lu b le n itr o g e n pools must have occurred during the d i f ­ f e r e n t ia t io n p rocess . 89 DISCUSSION r^-e G lucosam ine-6 -phosphate S y n th e siz in g Enzyme in B la s t o c la d ie lla ' 111"* 1 ' * —■■'—" ■■ ■ -rl r~ ~T^~. - r~~ As a r e s u lt o f p u r if ic a tio n s tu d ie s i t was e sta b lis h e d th a t the GA.-6-P s y n th e s iz in g enzyme in B la s t o c la d ie lla was very s im ila r to the glu tam in e-F -6-P transam idase o f N eurospora. However, th e somewhat anomalous r e s u lt s obtained in i t s pH curve m erit fu r th e r comment ( c . f . Fig. 5). At f i r s t glance i t might be presumed th a t the r e la t iv e in c r e a s e in a c t i v i t y w ith G-6-.P a t h igh pH, i t s decrease at low pH, was due to th e presence o f th e phosphoglucose isom erase. The l a t t e r , whose pH optimum is around 9 . 0 , lo s e s b e tte r than 90 $ o f i t s a c t i v i t y a t pH 5»0 i^Slein, 1955)* The weak a c t iv i t y of th e isom erase would indeed by expected to d ecrease th e o v e r a ll ra te o f low pH, and by the same to k en , i t would e x p la in th e s lig h t in crea se in a c t i v i t y w ith F-6-P a t the same pH, i . e . , l i t t l e would be removed by iso m e riz a tio n to G-6-P. At pH 8.5> however, th e G-6-P a c t i v i t y exceeds th at w ith F -6 -P 'a s the s u b s tr a te and t h is could h ard ly be due to isom erase a lo n e . In r e a c tio n m ixtures in which on ly F-6-P was present at the s t a r t , the co n cen tra tio n o f th e fr u c to s e isomer would always be high er than in th o se where i t was b ein g produced from 0-6-P v ia the isom erase. S in c e -th e u n fr a ctio n a te d supernatant was used in th e s e experim ents, i t appears p o s s ib le th a t a second enzyme system in the crude homogenate u t i l i z e d G-6-P in the same manner as the l i v e r enzyme d escrib ed by P o g e ll and Gryder.(1 9 ^ J) • The c a p a c ity o f G-6-P to e lu te enzymatic a c t i v i t y from the g e l preparations (in th is respect even su p erior to 90 th e s y n th e tic F-6-P c o n ta in in g no G-6-P) provides some support fo r th e id e a o f a second enzyme. th e r e a c tio n The f a c t th a t the ca p a city o f G-6-P to promote d ecrea ses during R. S. developm ent, and. th a t th is cannot be ex p la in ed by decreased isom erase a c tio n a ls o su g g ests a second pathway to GA-6-P v ia t h is su g a r. Only sep a ra tio n of the two ( i f th ere are tw o) -types o f a c t i v i t y from each o th er, and from th e isom erase, w i l l remove th e problem from th e area of s p e c u la tio n where i t now r e s ts s e c u r e ly . Growth vs . D if f e r e n t ia t io n in B la sto c 1a d ie l1a The Point o f No R eturn.— The. photographic record and th e l i n e a r i t y o f th e lo g a r ith m ic growth curves during the f i r s t 30 hours s u g g e st, f i r s t , th a t th e p lan ts are reasonably w e ll synchronized and, second, th a t th e r a te o f growth i s constant during th is p erio d . Up to th e age o f 30 hours th e p lan ts appear id e n t ic a l w ith 0 .C . t h a l l i at a comparable sta g e in t h e ir gen eration tim e . The f i r s t m orphological in d ic a tio n of development toward R. S. p la n ts can be seen ( c . f . F ig . 9 , 30 h r . ) as an accum ulation o f cytoplasm ic m a teria l in the term inal p o rtio n of the t h a l l u s , i . e . , th e region d e stin e d to become the r e s is ta n t sporangium. Although th e lo g a rith m ic curves fo r th e parameters in v estig a ted " during th is stud y are not a l l p r e c is e ly lin e a r fo r the period from 0 to 30 h o u rs, th e average r a te s ( i . e . , s lo p e s ) are a c tu a lly q u ite s im ila r to th o se f o r th e dry w eight and volume in c r e a s e s . Thus, in s p it e o f some s lig h t d iffe r e n c e s in th e r a te s fo r in d iv id u a l parameters at d i f ­ feren t' p e r io d s, t h is made i t d i f f i c u l t to d e te c t any e f f e c t s a sso c ia te d 91 w ith d i f f e r e n t ia t io n rath er than growth per s e . For example, the maximal r a te f o r c h it i n d e p o s itio n per plant occurs from 0 to 12 hours, th a t fo r glucosam ine sy n th e ta se and glu cose-6-p h osp h ate dehydrogenase a c t i v i t y as w e l l as s y n th e s is o f n o n -p ro tein s o lu b le n itro g en ^SNPN) and so lu b le p r o te in n itr o g e n (SPN), between 12. and 2 k hou rs, and th at fo r melanin d e p o s itio n from 2 k to 36 hours . But, ir r e s p e c t iv e o f th ese s lig h t changes, th e q u a n titie s and a c t i v i t i e s per p lan t o f each o f the above, as w e ll as t o t a l n itro g e n (TN), l i p i d , e t c . , in crea ses s t e a d ily during th e p eriod o f lin e a r growth. The. c a p a c ity o f such p la n ts to rev ert to an O.C. type when th ey are removed from th e b ica rb o n a te, provided th ey are not more than _ca. 36 hours o ld , proves th at no ir r e v e r s ib le changes e s s e n t ia l to th e str u c tu r e and fu n c tio n o f an R. S. plant have occurred up to th a t s ta g e . By the same to k en , i t can be assumed th at' any changes occurring th e r e a fte r are d e f i n i t e l y a s so c ia te d w ith R. S. d if f e r e n t ia t io n . Whatever th e s p e c if ic p r o c esses may be which cause form ation o f the septum and c e s s a tio n o f growth, th ey have a profound e f f e c t upon the whole c e l l which appears to reach i t s clim ax a t 36 hours-. On to g e n e tic Changes on a Per Plant B a sis .--The v a lu e o f ex p ressin g d a ta in terms o f the in d iv id u a l p lan t cannot be overemphasized when co n sid e r in g th e problem o f growth v s . d if f e r e n t ia t io n , This approach has been found u s e fu l in stu d y in g th e development o f such d iv e r se organisms as amoebae (P r e s c o tt, 1 9 5 5 ), and higher p la n ts ^Brown and Robinson, 1955)- In B la s t o c l a d ie lla , th e a c t iv it y o f glucosamine 92 sy n th e ta se per gram dry w eight reaches a maximum at approxim ately 2l± h o u rs, y e t th e a c t i v i t y per u n it p ro tein f a i l s to reach i t s peak u n t i l about I48 h o u rs. However, i t can be. seen th at the t o t a l a c t iv it y per p la n t a tt a in s i t s apex at 36 hou rs, then d e c lin e s ( c . f . F ig s . 1 2 - lU ) . Two im portant c o n c lu sio n s can be drawn from th ese f a c t s : 1) th e 2.U hour peak,J per u n it dry w e ig h t, r e s u lt s from a more rapid sy n th e sis o f th e enzyme than o f oth er p ro tein c o n s titu e n ts ( c . f . lo g p lo t in F ig . 1 )|B fo r in c r ea se d slope),* 2) the in creased s p e c i f ic a c t i v i t y r e f l e c t s a d ecrea se in oth er s o lu b le (enzym atic ?) p ro tein s a ft e r 36 hours, rath er than a n e t s y n th e s is o f glucosam ine sy n th e ta s e . This o b serv a tio n i s im portant because i t lea d s to th e conclusion, th a t th e r e te n tio n o f t h is enzyme i s required fo r the continued sy n th e sis o f c h it in in th e R.S. a f t e r 36 h o u rs. The sy n th eta se reaches i t s maximum q u a n tity per c e l l at th e sta g e when approxim ately S0% o f the t o t a l c h it i n is s t i l l to be form ed. The behavior o f th e sy n th eta se when G-6-P i s used as su b str a te is more d i f f i c u l t to in t e r p r e t . The peak in s p e c i f ic a c t i v i t y a t '2.1 to 22 hours may m irror th e h ig h e s t l e v e l o f isom erase a c t iv it y reached at t h is age ( c . f . F ig . 2U) • On both per w eight and per p lan t b a se s, maximum s y n th e s is o f GA-6-P from G-6-P occurs at the same age as does th a t from F -6-P . The p o s s i b i l i t y th a t the decrease in G-6-P u t i l i z a t i o n r e la t iv e to F-6-P a f t e r 36 hours was due to the d e c lin in g a c t i v i t y o f a second 0-6-P s p e c i f i c enzyme has alread y been m entioned. There i s , p r e se n t, i n s u f f ic ie n t in form ation to warrant a d e f in it iv e ex p la n a tio n o f t h is phenomenon. 93 I t .is in t e r e s t in g to note that w h ile both c h it in con ten t ( c . f . Table XVI) and maximal s p e c if i c a c t i v i t y (1. 29 jaM/mg. p r o te in /2 0 m in.) * o f th e sy n th e ta se in c r e a se s when p la n ts are grown in the h ig h er bicarb on ate concen tration* th e amount o f enzyme per gram dry weight does n o t. Although p e r -p i ant data were not obtained fo r th is p a r tic u la r phase o f th e work, i t can be in fer r ed th a t th e changes are th e r e s u lt o f in crea sed u t i l i z a t i o n by p r e fe r e n tia l d iv e r sio n o f su b str a te s through t h a t s y n th e tic pathway. The le v e lin g o f f o f the T P N -specific g lu c o s e -6 - phosphate dehydro­ genase a c t i v i t y at 3.6 hou rs, fo llo w ed by a f a i r l y la r g e in c r ea se again a f t e r 60 hou rs, im p lies th a t during R. S. d if f e r e n t ia t io n th is system tak es on added s ig n if ic a n c e in c e ll u la r energy metabolism or the pro­ d u ctio n o f e s s e n t ia l m e ta b o lite s . Both T P N -specific glu cose-6-p h osp h ate dehydrogenase and 6 - phosphogluconic dehydrogenase a c t i v i t y are presen t in mature 0 .C . p la n ts (Cantino and H o ren stein , 1939) which have an endogenous Qq ^ o f a6out 10 to 30 (McCurdy, 1959) • On th e other hand, th e s h i f t toward a t y p ic a l ly o x id a tiv e pathway during d if f e r e n t ia t io n o f R. S. p la n ts , suggested by th e in crea se in g lu cose-6-p h osp h aie dehydrogenase, i s somewhat d i f f i c u l t to r e c o n c ile w ith the g r e a tly reduced Qo_ ( 0 . 1 ) -of the mature R. S. plant (C antino, L o v ett, and H o ren stein , 1957)* The lack o f cytochrome oxidase in mature R. S. p la n ts (C antino and H o ren stein , 1955) precludes th e use o f reduced TPN v ia a term in al o x id a se . Presumably i t would a lso not be in volved in the p olyp henol o xid ase system (which can be coupled to TPN s p e c if ic r ed u c tio n sj Cantino and H oren stein , 1955) sin c e no n et sy n th e sis of 9U m elanin occurs a lt e r 60 h o u r s. However, one obvious p o s s i b i l i t y would be i t s co u p lin g w ith th e r e d u c tiv e ca rb o x y la tio n o f a - k e t o g lu ta r a te . The a c tu a l amount o f reduced TPN produced in th e maturing R. S. p la n ts and the ex ten t o f i t s u t i l i z a t i o n in s y n th e tic r e a c tio n s during ontogeny are a t p resen t unknown, but i t would be an in t e r e s t in g area fo r fu tu r e in v es t i gat i o n . The bimodal aspect o f th e r e la tio n s h ip between age o f t h a l l i and t h e ir c h it in co n ten t ( c . f . Fi g. 18) apparently r e s u lt s from a h igh er r a te o f c h it i n s y n t h e s is , as compared to other c e l l c o n s titu e n ts , during th e f i r s t 12 hours ( c . f . r a te s in F ig . 19)* E x a ctly the o p p o site r e la t io n s h ip occurs' during the period from 12 to 36 hou rs. Y et, i t became a sim ple m atter to in te r p r e t t h is apparently anomalous- behavior when it- was found th a t most o f the oth er parameters of growth in crea se most s t r ik in g l y ( c . f . lo g p lo t s ) from 12 to 30 hours w h ile th e r a te of c h i t i n s y n th e s is a c tu a lly d e c r e a s e s . C h itin d e p o s itio n l i e s a t the o p p o site end o f the b io s y n th e tic sequence which b egin s w ith the glucosamine sy n th eta se rea c tio n j at f i r s t g la n c e , c h it i n sy n th e sis might be expected to mimic the changes in a c t i v i t y o f the s y n th e ta s e . However, Changes in any o f th e in t e r ­ m ediate ste p s co u ld , and probably do, a lt e r t h is r e la t io n s h ip . Furthermore, i t i s obvious th a t the ra te o f c h itin sy n th e s is would a ls o be dependent upon th e a v a il a b i l i t y o f hexose-phosphates and com p etition by oth er system s req u irin g th e same s u b s tr a te s . Such an in te r p r e ta tio n may be invoked to e x p la in th e d iscrepan cy between the maximal r a te s f o r c h it i n accum ulation from 0 to 12 hours, and fo r glucosamine sy n th eta se 95 from 12 to 21; hours . These r e s u lt s make i t m a n ife stly c le a r that the s p e c i f i c a c t i v i t y o f an enzyme per se o n ly in d ic a te s i t s p o te n tia l and cannot y ie ld more than a p a r t ia l understanding o f the a c tu a l r a te of th e r e a c tio n in v i v o . In f a c t the d if fe r e n t methods o f d e fin in g s p e c i f i c a c t i v i t y , e . g . , on th e bases o f dry w eig h t, n itr o g e n , p r o te in , e t c . , o fte n ser v e to confu se rather than c l a r i f y th e s it u a t io n . But, n o tw ith sta n d in g the d i f f i c u l t i e s in in te r p r e tin g i t s enzymol o g i c a l b a s is , i t should be noted in co n clu sio n th a t w h ile c h it in accum ulation accounts fo r approxim ately. 21$ o f th e in c r ea se in dry w eight a f t e r 36 hou rs, i t co n trib u tes only about 11$ to th e f i n a l dry w eight o f th e in d iv id u a l mature plant . The s y n th e s is o f l i p i d e x a c tly p a r a lle ls the in crea se in dry weight " (logarith m ic p lo t s ) and reaches i t s maximum q u a n tity at I48 hours ( c . f . F ig . 2 3 ) . It i s again in t e r e s t in g to observe th a t l i p i d s y n th e sis alon e accounts f o r . 21;$ o f th e in c r ea se in dry w eight from 36 to I4 .8 h o u r s , and c o n tr ib u te s alm ost 20$ o f th e w eight a t t h is age. The sub­ seq u en t d ecrease in f a t from Ij.8 to 83 hours accounts fo r 50$ of the d e c re a se in dry weight which occurs during t h i s in t e r v a l. The s i g n i f i ­ cance o f th e change is unknown, but i t i s p o ssib le th a t i t i s re­ u t i l i z e d f o r s y n th e tic p r o c e sse s, or perhaps converted to forms not r e a d ily e x tr a c ta b le . The d ecrease is co rrela ted w ith the appearance, by 83 h ou rs, o f the f a i r l y la r g e , d i s t i n c t " lip id ic globules'* ch aracter­ i s t i c o f th e cytnplasm o f mature R.S. (F ig . ? , 83 h r . ) . In any ev en t, c h it i n and l i p i d to g e th er make up almost on e-th ird o f th e t o t a l dry m a ter ia l in mature p la n ts . The magnitude of the f a t accum ulation lead s 96 ■to th e s u p p o sitio n th a t th is i s fo r the ’’purpose’’ o f e f f i c i e n t energy s to r a g e s in c e R .S . can remain v ia b le fo r s e v e r a l years (C antino, unpub­ lis h e d ) . The d is r u p tio n o f th e c i t r i c acid c y c le by bicarbonate (Cantino and H y a tt, 1953c) provides a p o s s ib le ex p la n a tio n fo r the in d u ctio n o f in c r ea se d f a t s y n th e s is in th e develop in g R .S. p la n t. I f a c e t y l- coenzyme-A was unable to en ter the c y c le by way o f the condensing enzyme system , i t s accum ulation could lea d to the increased s y n th e s is o f f a t t y a cid s v ia th e con ven tion al pathway. The sy n th esis' o f over 90% o f th e f a t t y a cid carbon from a c e ta te has been shown by O ttke, e t a l . (1 9 5 l) 1 f o r N eurospora, and fo r y e a st by "White and Werkman (19U7) • Of even g r ea ter in t e r e s t are th e rep orts o f bicarb on ate- and reduced TPNdependent system s fo r the sy n th e sis o f ' lon g chain f a t t y a cid s from acetyl-coenzym e-A in pigeon li v e r (G ibson, T itch en er, and W akil, 1958) and avocado (S q u ir e s, Stumpf, and Schm idt, 1 9 5 8 ). The in tr ig u in g p o s s i b i l i t i e s o f such a sy n th e s is in r e la t io n to the bicarbonate e f f e c t in B la s t o c l a d ie lla are ob vio u s, p a r tic u la r ly in view o f th e b u ild -u p o f an a c tiv e zwischenferment which could provide th e n ecessa ry pool o f reduced TPN in th e d evelop in g R .S. - The s y n th e s is o f y -c a r o te n e i s c h a r a c te r is tic of th e R .S. sin c e i t s p resence cannot be d e te c ted in the normal mature 0 .C. p lan ts (Cantino and H o ren stein , 1 9 5 6 ). The fu n c tio n of th is pigment i s un­ known although i t i s produced by a very few ( l e s s than 2%) th in -w a lle d p la n ts o f B l a s t o c l a d ie lla , and occurs in th e male gametes o f the c l o s e l y r e la t e d genus A llom yces♦ Although the tim e course fo r ^ -ca ro ten e prod u ction was not e sta b lis h e d q u a n tit a tiv e ly , i t appeared from v is u a l in s p e c tio n to roughly p a r a lle l th e in crea se in lip id during growth of P. .S . p la n t s . The form ation o f carotene in Mucor h e im a lis in v o lv e s th e u t i l i z a t i o n o f a c e ta te carbon (Grob and B u tle r , 19£ 6 ) . This pre­ sumably •occurs v i a th e form ation o f mevalonic or d im eth y la c ry lic acid from acetyl-coen zym e-A , follow ed by p olym erization in to la r g er is o pre.no id u n its . Such a s y n th e tic sequence in Bla s toe lad i e l l a could be favored by th e backing up of a c e ta te , or acetyl-coenzym e-A , in the same fa s h io n as was suggested fo r induced f a t production. Evidence th a t th is may be so stems from th e ob serv a tio n s (Cantino and H yatt, ■1953b) th a t a mutant o f B la s t o c la d ie lla w ith a le s i o n in th e Krebs c y c le accumulates la r g e q u a n titie s o f V -c a r o te n e . A fu n c tio n a l polyphenol oxidase system in mature R .S . p lan ts and i t s c o u p lin g (e s ta b lis h e d in v i t r o ) w ith the red u ctiv e ca rb o x y la tio n o f a -k e to g lu ta r a te h a s 'been d escrib ed by Cantino and H orenstein (1955)* Tn th e p resent study i t was determined th a t the in vivo d e p o s itio n o f m elanin occurs most r a p id ly during th e period from 2k to 36 hours, but th e age at which th e polyphenol oxidase f i r s t appears is s t i l l unknown. N itrogen Metabolism and D if f e r e n t ia t io n .—The in crea se in t o t a l n itr o g e n per p lan t (a s a lo g a rith m ic fu n c tio n ) c lo s e ly p a r a lle ls th a t f o r i t s dry w eight ( c . f . F ig s . 25 and 1.1). On the oth er hand, a d ecrea se in s o lu b le n itro g e n and an in c r ea se in t o t a l n itro g en per u n it dry w eight occurs during th e f i r s t 12 hours o f growth. The d iffe r e n c e between the s o lu b le and t o t a l q u a n titie s in swarmers ( c a . 25 Jig./m gdry w eigh t) i s o f th e same order o f magnitude as the n u c le ic acid 98 n itr o g e n at t h is sta g e [ Turian and Cantino, i 9 6 0 ) . B efore the swarmers germ inate, th e s e n u c le ic , a cid s are a sso c ia te d almost e n t ir e ly w ith two d i s t i n c t s tr u c tu r a l u n it s , th e nucleus (D N A ), and the n u clear cap vE-NA) w hich would be expected to sedim ent w ith the c e ll u la r d eb ris upon c e n tr ifu g a t io n . During the f i r s t 12 hours o f growth, th e in crea se in t o t a l n itro g e n i s la r g e l y due to c h it i n s y n t h e s is . On the oth er hand, th e apparent d ecrea se in s o lu b le p r o te in per m illigram dry w eight during t h is period may be an a r t if a c t due to the in co rp o ra tio n o f p ro tein in t o the r a p id ly expanding w a ll and r h iz o id a l system o f th e young p la n ts . P lan ts homogen­ iz ed at 12 hours do not fragment but m erely crack open to r e le a s e t h e ir p r o to p la s t s . The shapes o f th e sporangia and r h iz o id s remain remark­ ab ly .in t a c t and i t i s p o ssib le th a t the p ro tein s in the r h iz o id s and some o f th o se :in or on th e sp o ra n g ia l w a ll were not reco v ered . A d ecrea se in th e fr e e amino acid pools a ls o occurs in th e f i r s t few hours and i t i s assumed th a t th is r e s u lt s from both u i t l i z a t i o n and d ilu t io n due to rapid growth. A most S tr ik in g phenomenon i s the rapid in c r e a se in the so lu b le n o n -p ro tein n itro g e n (SNPN) between 2 k and 36 hours ( c . f . F ig s . 26 and 2 7 ). The corresponding in c r e a se in th e fr e e amino acid s dem onstrates th a t the r is e is la r g e ly due to changes in th e s e p o o ls . On a dry w eight b a s is , th e SNPN doubles during t h is in t e r v a l w h ile th e so lu b le p r o te in n itro g en ( S P N ) d e c r e a se s. lThe swarmers contained c a . 203 to 287 pg- t o t a l n u c le ic acid/m g. dry w eight.. I f a n itr o g e n con ten t o f 16$' is- assumed fo r B la s t o c la d ie lla n u c le ic a cid ^based on y e a st d a ta ), th e above q u an tity of. n u c le ic acid would c o n tr ib u te c a . 32 to U6 pg n itrogen/m g. dry weight . 99 Once a g a in , however, th e changes are more e a s i l y understood when con­ sid e re d on a per p lan t b a s is . This approaoh r e v e a ls th a t a d i f f e r e n t i a l in c r e a se in amino a c id s v s . p ro tein occu rs, the process reaching i t s clim ax a t 36 h o u r s. At t h is sta g e the SNPN exceeds the SPN by about 3 b to 35$* and th e two to g e th er comprise a l l but a sm all fr a c tio n ( 6 %) o f th e .t o t a l n o n -ch itin o u s n itro g e n o f the p la n t. The la r g e change in th e fr e e amino acid pools co u ld , o f co u rse, r e s u lt from uptake o f n itro g e n from th e medium, th e breakdown and r e o r g a n iz a tio n o f p r o te in s , or b oth . That the two p rocesses may be in v o lv ed i s s tr o n g ly su ggested by the r e s u lt s o f e le c tr o p h o r e tic stu d ie s (C an tin o, L o v e tt, and H oren stein , 1937); th ree out o f four s o lu b le p r o te in fr a c tio n s in O.C. p lan ts were absent in R .S. p la n ts , w h ile the l a t t e r contained a s o lu b le p ro tein not found in O.C. p la n t s . The re­ v e r s i b i l i t y o f th e system up to approxim ately 36 hours would suggest th a t th e s e changes must occur e ith e r during (o r a ft e r ) t h is tim e, or th ey must be e a s i l y and r a p id ly r e v e r s ib le . f a ilu r e to d e te c t an in crea se in the s o lu b le p ro tein s from 36 to H8 h o u rs, concom itant w ith the $0% decrease in SNPN, c le a r ly im p lies con version o f th e l a t t e r to c h it i n , in s o lu b le p r o te in , and/or purines * and pyrim idines and n u c le ic a c id s . Although in s u f f ic ie n t data are a v a ila b le , c e r ta in general co n clu sio n s can be drawn concerning the f a t e o f t h is fr a c tio n * At the very m ost, the in crea se in c h it i n during th is period could o n ly account fo r 26% o f the decrease in SNPN. Also during the same tim e in t e r v a l, the in crea se in t o t a l n itro g en i s 1 .U tim es the d ecrease in SNPN, in d ic a tin g th at n itro g en uptake i s s t i l l o ccu rrin g . 100 This o b v ia te s any n ecessa ry c o r r e la tio n between the in c r ea se in c h it i n and th e d ecrease in th e SNPN p o o ls . It d o es, however, mean that th ese s o lu b le p ools are being u t il iz e d more ra p id ly -th a n th ey are being r ep len ish e d . F in a lly , c o n sid e r a tio n of the u ltim a te fu n c tio n o f a mature R.S. perm its an educated guess concerning the form o f the in s o lu b le , nonc h itin o u s n itr o g e n compounds produced during the 36 to I48 hour p erio d . The R .S . plan t e v e n tu a lly uses e s s e n t ia ll y a l l o f i t s protoplasm ic co n ten ts to produce m o tile swarmers. I f the in s o lu b le , n o n -ch itin o u s n itr o g e n i s c a lc u la te d as the equivalen t amount o f n u c le ic acid per m illigram dry w eight o f protoplasm (se e fo o tn o te on p. 9 8 ) , a value o f about 200 pg i s o b ta in e d . This i s e x a c tly the order of magnitude actu­ a l l y obtained by a n a ly s is o f swarmers, a remarkable c o r r e la tio n indeed I Thus, th e dat a su ggest th a t a co n sid era b le proportion of the. SNPN i s used fo r th e s y n th e s is o f n u c le ic a c id s . The su b ject m atter ju s t d iscu ssed provides some exp lan ation o f the changes in the o v e r a ll l e v e l o f the fr e e amino acid p ool during the growth and d if f e r e n t ia t io n o f an R.S. p la n t. It is more d i f f i c u l t to a sc r ib e a r o le to the changes which p a r tic u la r amino acid s undergo. The almost com plete disappearance o f ty r o sin e v.an e sta b lish e d su b str a te f o r th e polyphenol oxid ase in B la s t o c la d ie lla ) aft er 3 6 . hours, may w e ll be due to i t s rapid o x id a tio n fo r melanin s y n th e s is . The r e la t iv e l y la r g e a la n in e , and sm all glutam ate, pools at U8 hours correspond to the peaks in both i s o c i t r i t a s e and g ly o x a la te -a la n in e transam inase, a c t i v i t i e s which occur at the same tim e (McCurdy, 1959). and which are known 101 to provide a p o te n tia l pool o f g ly c in e fo r b io s y n th e sis in B l a s t o c l a d i e lla . t h is i s c o n s is te n t w ith the -notion proposed above th at n u c le ic acid s y n t h e s is , and th e r e fo r e purine and pyrim idine s y n th e s is , occur a fte r 36 h o u rs. Glutamate could play a tw o -fo ld -role in the system: fir s t, as th e sou rce o f th e ala n in e amino group by transam ination w ith pyruvate (McCurdy, 1999).; and second, by am idation, as th e source o f th e glutam ine required fo r purine s y n th e s is . A sp artic a c id , which a ls o d ecreases sh arp ly a f t e r 36 hou rs, is in v o lv ed in the sy n th e sis of both purines and pyrim idines in oth er organisms and might play a sim ila r r o le in B la s to c la d ie lla . The d isp rop ort ion ate in crea se in th e glutamate pool at 83 hours is in t e r e s t in g b u t, for th e time b ein g , in e x p lic a b le . I t i s o f course p o s s ib le that, th e in crea se is an in d ic a tio n of i t s act i v i t y in various tra n sa m in a tio n s, i . e . , th at i t serves as a n itro g en b o t t le neck or fu n n el fo r n itro g e n tran sform a tio n s, and that i t in c r ea se s in concen­ t r a t io n when no lo n g er a c t iv e ly u t i l i z e d . A rather unique r o le fo r glutam ate in B la s t o c la d ie lla was e a r lie r suggested by the fa c t th a t i t was th e only amino acid ab le to serve as the primary source o f n itro g en in a s y n th e tic medium (Barner and C antino, 19 9 2 ). The in v iv o sy n th e sis o f glutam ate from glucose-U -C 14 w ithout apparent m ediation o f th e c i t r i c acid c y c le , and i t s slow e q u ilib r a tio n w ith a -k e to g lu ta r a te (Cantino and H o ren stein , 1996) a ls o suggest th a t t h is amino a cid d isp la y s unusual behavior in B l a s t o c l a d i e l l a . General Conclusio n s .— The process o f R. 3 . development can be summarized as fo llo w s : T h e -fir st observable e f f e c t o f th e bicarbonate 102 in d u cin g agent i s a reduced growth r a te , presumably by a d isr u p tio n o f the. o x id a tiv e m etabolism o f the c i t r i c acid c y c le and i t s a sso c ia te d cytochrome system . With th is excep tion no other changes are d etected u n t i l 2lj. to 36 hours a fte r spore germ ination. At about 30 hours the m o rp h o lo g ica lly d is tin g u is h a b le m igration of' cytoplasm toward th e s i t e o f th e fu tu r e sporangium o c cu rs. This i s follow ed by changes in enzyme s y n th e s is and oth er c e llu la r p rocesses in d ic a tin g a fundamental re­ o r g a n isa tio n w ith in the c e l l . The culm ination o f th ese p rocesses by c a . 36 hours i s accompanied by the ir r e v e r s ib le commission of th e plant toward R .S . form ation . F ollow ing th is s ta g e , a period b est d escrib ed as the m aturation phase en su es, during which fu rth er changes in enzymes and sto r a g e products occur. The r e s u lt is . the round, m elanin-pigm ented, th ic k -w a lle d , l i p i d - and Y -c a r o tin e -c o n ta in in g R .S ., la ck in g cytochrome o x id a se as w e ll as most o f th e c i t r i c acid c y c le enzymes, and d is p la y ­ in g a v ery low r e s p ir a to r y a c t i v i t y . This stru ctu re surmounts an e s s e n t i a l l y empty-and l i f e l e s s low er s t a l k . As might perhaps be exp ected , each new b it o f inform ation gained in a stud y of th is kind r a is e s more new q u estion s than i t answers o ld . However, 'at each sta g e o f our accum ulating knowledge we are in a p o s itio n to frame th e se q u estion s w ith in c r e a sin g s o p h is tic a tio n and p r e c is io n . Probably th e most fundamental q u estio n posed by our present knowl­ edge i s th e foIT owing: s in c e .it i s known th at the process le a d in g to R .S . form ation becomes in c r e a s in g ly ir r e v e r s ib le from- about 2h to 36 h ou rs, what system or system s are r e s p o n sib le , how are th ey formed, and at what sta g e do th ey begin to evolve toward th is con d ition ? Xt w i l l 103 o b v io u sly be im p o ssib le to com pletely answer t h is q u estio n fo r some tim e , but i t i s in s tr u c tiv e to consider the p o s s ib le mechanisms by which such a s it u a t io n may come to e x is t . W eiss (19U9) has suggested th a t a c e l l could be considered s t a t i s ­ t i c a l l y in terms o f the '"ecology o f an organized m olecular p o p u la tio n ,” w ith a l l th e in te r a c tio n s and interdepend en cies which the term eco lo g y im p lie s . I f such a concept is accepted , growth could be defin ed in W eiss 5s terms ”as the in c r ea se in th e s iz e of a given population w ithout e s s e n t ia l change in ch aracter ''; and, '" d iffe r e n tia tio n , as a p ro g ressiv e change in th e com position of th e m olecular population in clu d in g the appearance o f new, and th e disappearance o f o ld , sp e c ie s Such a d e f in it io n n e c e s s a r ily im p lies th a t any m orphological changes must be preceded by d if f e r e n t ia t io n at th e m olecular l e v e l . In th e in sta n c e o f B la s t o c la d ie lla the f i r s t m orph ologically d e te c ta b le d if f e r e n t ia t io n i s the cytoplasm ic m igration at 30 hou rs. I t must be assumed that some m olecular changes had already occurred p reviou s to th a t time . The only o b v io u sly new parameter so fa r de­ m onstrable b e fo r e 30 hours i s a s lig h t amount o f melanin sy n th e sis b egin n in g at about 21; h o u rs. However, it i s known th a t t h is process can be uncoupled from P..S. d if f e r e n t ia t io n without otherw ise e s s e n t i a ll y a lt e r in g i t s progress (C antino, 1953)- T e n ta tiv e ly , th e r e fo r e , i t can be presumed th at i t i s n o t, o f i t s e l f , important in causing th e plant to d i f f e r e n t i a t e . The fact, th a t melano genes is i s , n e v e r th e le s s , a c h a r a c te r is t i c o f the P, .3 * developm ental sequence, leads to the conclu— 2 ion th at i t s i n i t i a t i o n must r e s u lt from even e a r li e r subm icroscopic 10h even ts . thus i t can be s ta te d w ith reasonable c e r ta in ty that some change, dr changes, in volved in P .S . d if f e r e n t ia t io n must occur before 2 U hours o f a ge, although no time lim it can be placed as to the e a r l i e s t a lt erat i o n . Having at l e a s t narrowed down the l i m i t s , our a tte n tio n can be addressed to the f i r s t part o f the q u estio n concerning the system s in ­ volved and how th ey came to e x i s t . With the lim ite d knowledge at our d is p o s a l t h is can, at b e s t, o n ly be done in very general term s. At le a s t one primary e f f e c t of bicarbonate has already been d e scr ib e d , i . e . , the op eration o f th e SKI c y c le a n d 'd isru p tion o f the normal c i t r i c a cid c y c le . How, th en , can t h is lea d to the d if f e r e n t ia t io n o f an R .S. p lan t? " E s s e n tia lly two p o s s i b i l i t i e s e x i s t , both based on the assump­ t io n that the bicarbonate mechanism r e s u lts in the p ilin g up o f in t e r m ediatesj i t has alread y been demonstrated fo r a -k e to g lu ta r a te th a t such p il in g up does in f a c t o ccu r.' The f i r s t is that th ere is an in ­ creased u t i l i z a t i o n o f s p e c if ic p r e -e x is t in g , or c o n s t it u t iv e , enzymatic pathways le a d in g to an o v e r a ll q u a n tita tiv e s h i f t in the in t r a c e llu la r b a la n c e . The second i s that in a d d itio n to the c o n s titu tiv e system s, new and unique enzymatic sequences may be induced, and c e r ta in old ones e lim in a te d , ' by the presence o f increased le v e l s o f endogenously produced in term ed ia tes • The evid en ce to date str o n g ly su g g ests that the second a lte r n a tiv e more adequ ately d e scr ib e s the s itu a t io n in B la s t o c l a d ie lla . At le a s t one enzyme system , the polyphenol o x id a se , i s formed de novo during r «S. developm ent, and the form ation of a second, fo r "jr—carotene 105 s y n t h e s is , i s probab le. Other pathways which are known to be a lt e r e d y or could p o s s ib ly be a lte r e d , by in t r a c e llu la r in term ed iates have alrea d y been m entioned. The r e v e r s i b i l i t y o f the B la s t o c la d ie lla system up to a c r i t i c a l point appears to d is tin g u is h i t from th at in b a c te r ia where an enzyme once induced i s not broken down again but merely d ilu te d out o f e x is te n c e by subsequent growth and reproduction (Hogness, Cohn, and Monod, 1 9 5 5 ). The in t r a c e llu la r changes in t h is organism appear to be much more akin to the s t a t e o f ’ "dynamic equalibrium" found fo r mammalian c e l l s (Heimberg and V e lick , 195h; V elick 1 9 5 6 ). This suppo­ s i t i o n i s supported by the complete lo s s o f se v e r a l c o n s titu tiv e enzymes, and the changes in the glucosamine sy n th e ta se , g lu c o s e -6 - phosphate dehydrogenase and isom erase enzymes ( c . f . F ig . ;2U0 - It should be noted w ith resp ect to Figure 21+ th a t a fte r 36 hours th ere is no in c r e a se in th e s o lu b le p r o te in , nor does growth occur a fte r 1+8 hours . D esp ite t h i s , th e l e v e l s o f the few examples stu d ied do change q u a n tita tiv e ly ; both in c r e a s e s and d ecreases occur, i . e . , th e p ro tein s are presumably 1 b ein g sy n th esiz ed a t c e r ta in periods and degraded at o th e r s. A p o rtio n o f the o r ig in a l q u estio n posed has been answered; part remains unanswered. However, though i t i s evident that th ere are la r g e gaps in our knowledge o f the c e l l u l a r p rocesses in B la s t o c l a d ie lla,; the concept o f a dynamic eq u ilib riu m , in flu en ced and rechanneled by the ^ e f f e c t s o f b ica rb o n a te, has proved u s e fu l as a working h y p o th e sis. -’■The argument th a t th e se changes could eq u a lly w e ll occur as the r e s u lt o f th e in h ib it io n , o r v ic e v e r s a , o f e x ist ing^ and p o t e n tia lly c a t a ly t ic p r o te in s cannot be ig n o r e d . This p o s s ib i lit y could only be elim in a ted by la b e lin g experim ents w ith is o to p ic tr a c er s during d i f ­ f e r e n t s ta g e s o f developm ent. 106 I t i s o n ly a lo g i c a l e x te n sio n of. t h is id ea to su ggest th a t th ese e f f e c t s lea d to an in c r e a s in g ly a lte r e d m etabolism , th e process eventu­ a l l y becoming a u to c a ta ly tic and ir r e v e r s ib le . The l a t t e r presumably occurs when a key in ter m e d ia te, or sy n th e tic pathway, reaches a c r i t i c a l th r e sh o ld and, by so d oin g, s h i f t s the whole equilibrium in a new d ir e c t io n . U n til such a th resh o ld i s reached, th e system could remain r e v e r s ib le by removal o f th e stim u lu s, i . e . , b ica rb o n a te. The technique described h e r ein fo r growing reasonably synchronized la r g e s c a le c u ltu r e s o f B la s t o c la d ie lla em ersonii has made f e a s ib le a d ir e c t approach to the problem during th e e a r ly c r i t i c a l periods o f grow th.' The r e s u lt s obtained by t h is technique have provided a frame o f r e fe r e n c e to ser v e as a guide in d evelopin g the experim ental ap­ proaches most l i k e l y to y ie ld answers to some o f the m anifold q u estion s a s s o c ia te d w ith morphogenesis in B la s t o c l a d ie lla . 107 SUMMARY 1 . The enzyme glucosam ine sy n th eta se (g lu ta m in e-F - 6-P transam idase) was p u r ifie d _ca 19- f o ld from e x tr a c ts o f B la s t o c la d ie lla emersonii and some o f i t s p ro p erties s tu d ie d . 2 . A method was developed which made p o s s ib le , fo r th e f i r s t time among th e Phycom ycetes, the study o f p h y sio lo g ic a l and m orphological p ro cesses during the w e ll synchronized growth of B ia s t o c la d ie lla from zoospores to mature r e s is ta n t sp oran gial p la n ts . 3 . The enzymes glucosamine sy n th e ta se , g lu c o s e - 6- phosphate dehydrogenase, and phosphoglucose isom erase, as w e ll as se v e r a l oth er nitrogenous and non -nitrogenou s c e ll u la r components were stu d ied during the course o f r e s is t a n t sp oran gial developm ent. 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I . 1956b. Deamination o f glucosam ine-6phosphate by crude and p u r ifie d c e l l - f r e e e x tr a c ts o f E. c o l i . B p c t. P r o c . 108. Wolfe,, J . B ., and Nakada, H. I . 1956c. Glucosamine degradation by E . c o fi.' I I . The isom eric conversion of glucosam ine-6-phosphate to frucotose-6-ph osp hat-e and ammonia. Arch. Biochem. B iophys. 61* : 1*8 9 - 1+97 • 115 Wolfe., J . B ., B r itto n , B . B . , and Nakada, H. I . 195?- Glucosamine d egrad ation by E. c o l i . I I I . I s o la t io n and s tu d ie s o f "phosphoglucosam inisom erase Arch. Biochem. B iophys. 66:333-339. W olfe, J . B ., M artinez, R. J . , and Nakada, H. I . 1959- The phosphoglucosam inisom erase r e a c tio n o f E sc h e r ich ia c o l i . Arch. Biochem. B iop h ys. 79-330-337W right, B. E ., and-Anderson, M. L. 1959* B iochem ical d if f e r e n t ia t io n in th e slim e mold. Biochim . B iophys. Acta 31*310-322. APPENDICES 116 APPENDIY I L is t o f A bbreviations AG-l-P ............. .. N -acetylglu cosam in e-l-p h osp h ate AG-6-P . . . . . . . . . . N -acetylg.lucosaroine-6-phosphate AG-1 , 6-DP ............. N -acetylglu cosam in e-1,6-d ip h osp h ate ATP ........................... A denosinetriphosphate BCP ......... ................. Bromcresol purple DNA ...................... D esoxyribose n u c le ic acid DPN ........................... Diphosphopyridine n u cle o tid e EDTA . . .................... E th y len ed ia m in etetra a cetic acid F-6-P ...................... Fructose-6-phosphate FDP . . . . . ............. .. F ru cto se-1 ,6 -d ip h o sp h a te G-1 ,6 - DP ................ G lu cose-1,6-diphosph ate G—6—P .................. .. G lu cose-6 - phosphate ' GA . . .........................Glucosamine GA-6-P .................... Glucosamine-6-phosphate GA-l-P .................... Glucosamine-1-phosphate P i ............................. Inorganic phosphate RNA ............. .. Ribose n u c le ic acid SPN ......... .................S o lu b le p ro tein n itro g en - SNPN ........................ S o lu b le non -p rotein n itro g en TCA ........................... T r ich lo ro a c e tic acid TN .................. TSN ...................... .. TPN T o t a l’n itro g e n T otal so lu b le n itro g en ......... ............ Triphosphopyridine n u cle o tid e UDPAG ...................... U ridinediphosphate N -acetylglucosam ine UDPGA ...................... U ridinediphosphate glucosamine 117 • APPENDIX II Two-dimensional Chromatographic Map o f Amino Acids Key 1 . L -alan in e 1 3 • L -leu cin e 2 . L -argin in e 1U- L -ly sin e 3 . L -a sp a r tic a c i d ■ 15 • DL-methionine U. D -glucosam ine 1 6. N -acetylglucosam ine 5- D -g lu c o se -6 - phosphate 1 7 . L -phenylalanine 6 . L -glutam ic acid 1 8 . L -prolin e 7 • L-glutam ine 19 • DL-serine 8 . L -g ly c in e 2 0 DL-threonine 9 . L -h is tid in e 21. L-tryptophan 1 0 . L -hydroxyproline 22. L -ty ro sin e 1 1 . Inorganic :phosphate 23 • D L-valine 12 . L -is o le u c in e 118 <--------- d 3 1 V M ' Q I 0 V O I N O I d O d d - I O N V i n a - ---------- LU l-l CL 119 £ ■od CD 1A a P f o I Pt CO I co i I QO I ftT\ a o CO I LPi 1_T\ £ P 0 P CD £ OS O o p «c\) Pi p C£ I I I •I CO o o H P II I I I I I ( a I ft 0 ® O -P i— i to Is I I O I cm r— r— c— I CO c o c\j u \ I I I 0 to Is CL. cO to o •P 0O •H to ft O0 £h P £i 0 u 0 p ■P •H cp O U \m 0\ Io o o rt ? 1 3 CD o I I I I I Pfc pf O 0 cO PQ ft .0 P 0 12 03 aO £ On • a -pt is I i—I I I I I ) I I i—i 1 NO o CP o 1 p f OJ P T P i I CM CM 0 -4 I— I 0 rH CM CM 1to o ft to 1 NO rH ft ,—O 0 CL. £ 0 0 •P to N -P ft 0 cO x< P 12 -P s > to "H § to s o o 1— GO NO NO 1 rH« rH• rH« NO H 1 • £ -p cO O w rP 1 w cO n o o rH o o ; m «3 to ~' T03 a! ON H I W -4 ra | On 1 1 1 1 CO NO NO i—! CM 1 1A1A 5 0 rN> CO o ■rH to •p ft CO CO 1 0 P 1 to to CONO to 0 1 NO Mp 4 to t i E to 0-. Mb ft «. 1 i 1 nO nO 0 nO nO to nO 1 i 0 CD I O CO O 1 1 a 1 ■=53 o 0 CO p P to IX, to o o CO to _d d 73 to & to P o A- GO 0 E 0 0 o 43 o ■P 0 O 0 0 •H Tb 1 0 O o 0 rP O 0 P O to 'H a E P 0 0 O P -H 0 0 _g 1 P H s CD O B ro s p 0 paper. 0 P