A STUDY OF THE RIBONUCLEEC ACID-FOLYPHGfiPI-{ATE COMPLEXES ISGLATED FROM AMBAENA VARRABILIS AND SYNCHRONIZED CHLORELLA FYfiEEVEOEBOSA Thesis for H19 Degree of DH. D. MICHEGAN STATE UNIVERSITY David L. Correll 1961 'gHESiS This is to certify that the thesis entitled A STUDY OF THE RIBONUCLEIC ACID-POLYPHOSPHATE COMPLEXES ISOLATED FROM ANABAENA VARIABILIS AND SYNCHRONIZED CHLORELLA PYRENOIDOSA presented by David L. Correll has been accepted towards fulfillment of the requirements for Ph.Do degree in Fisheries and Wildlife and Agricultural Chemistry \/ sji/xLCi: Qgtfi Ma'or rolessor 44. 5’, W DateWI 0-169 LIBRA R Y Michigan State University HWY 0? T car-MES I ETD BITCH nthouéh con gasphate has app “relic function or this study was 1111 m and physiolo fzesecorzplexee s zetabelic utilize PJJA-polyphos % which 11 cider carefully c Plexes were tract itzlexee were at 5'3 Other phoephai tebohydretee, 01 with curve in I % were 3' in effect 0: icliihOSPhate we: 3:? level and in» Wilt about 1111 11; 3“ cWiltione ABSTRAOT A STUDY'OF'THE RIBONUCLEIC ACID-POLYPHOSPHATE COMPLEXES ISOLATED FROM ANABAENA VARIABILIS AND SYNCHRONIZED CHLORELLA PYRENOIDOSA by David L. Correll Although considerable literature concerning poly- phosphate has appeared in recent years, no acceptable meta- bolic function of these compounds has been demonstrated. This study was undertaken to better characterize the struc- ture and physiology of the RNA-polyphosphate complexes. These complexes seemed to be a potential mechanism for the metabolic utilization of the polyphosphates. RNA-polyphosphates were isolated from Anabaena and Chlorella which had been raised in chemically defined media under carefully controlled conditions. The isolated com- plexes were fractionated on DEAE-cellulose columns. The complexes were shown to be free from serious contamination by other phosphate compounds, amino acids, protein, other carbohydrates, orIDNA. Changes in the complexes during the growth curve in Anabaena and the life cycle in synchronized Chlorella were studied. An effect of light on the ratio of total RNA to total polyphosphate was found. High light levels depressed the RNA level and increased the polyphosphate level. Low light brought about unusually high amounts of RNA. Under normal light conditions both organisms were found to have about 15 got-10168 or p riboseo This r barges in stadied in sync :::;lexea under enemas, and The compleJ tiaracteristics siphyeicel evi ind system and :3 the RNA to 1:} its found to be :erentrations s iistilled water Befcre dielys is iith toluidine t Emomced metac fitachl‘omatic re 3:5. mild acid c Yeast 13015”; 533531 817 ”l ‘3. 31“ Venom O . 0‘ kialy Zed corp “illness. R"a P01». 5311 deB‘Ge. '1‘} its L in“”Pl‘ettee David L. Correll micromoles of polyphosphate-phosphorus per micromole of RNA- ribose. This relationship was surprizingly consistent. Changes in six fractionated RNA-polyphosphates were studied in synchronized cultures of Chlorella. The six complexes underwent independent changes in amount, dialysis properties, and metachromatic character. The complexes isolated from Chlorella showed the same characteristics as those isolated from Anabaena. Chemical and physical evidence was obtained that both a hydrogen- bond system and covalent bonds are involved in the linkage of the RNA to the polyphOSphate. The hydrogen-bond system was found to be most stable at pH 7.h to 7.6 and at salt concentrations above 0.2 M. Exhaustive dialysis against distilled water resulted in the cleavage of this system. Before dialysis the metachromatic reaction of the complex with toluidine blue was very small. After dialysis a pronounced metachromatic reaction was produced. This metachromatic reaction was not released by boiling, freez- ing, mild acid or basic pH, EDTA, or RNase. Yeast polyphosphatase was not very active on undialyzed complex, but was very active on the dialyzed complex. RNase and snake venom phosphodiesterase both reduce the metachromasy of dialyzed complex. After short-time treatment with poly- phosphatase, RNase, or snake venom phosphodiesterase; the polyphosphate involved in the complex only dialyzed to a small degree. This,coupled with the low specific metachromasy, was interpretted as evidence that only short polyphosphate gins were left, :,:,:.jed t0 the RI?! Lie low molecule: The data obi citing a polyphc :2 its terminal : lithe "native" c Evolved in a hyn cf the REA . David L. Correll chains were left, but that these chains were covalently bonded to the RNA. This type of material also resembled the low molecular weight complexes, as isolated from Chlorella. The data obtained were consistent with a structure in- volving a polyphosphate of variable length, which had both of its terminal secondary hydroxyls esterified to RNA-ribose. In the "native" complex the polyphosphate chain might be involved in a hydrogen-bond system with the nitrogen bases of the RNA. A STUDY OF ti on com;Y E All) SY‘:I( 1" Part1 DepaPt! Depa: A STUDY OF THE RIBONUCLEIC ACID-POLYPHOSPHATE COMPLEXES ISOLATED FROM ANABAENA VARIABILIS AND SYNCHRONIZE) CHLORELLA PYRENODDOSA by David LE Correll A THESIS submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Fisheries and Wildlife and Department of Agricultural Chemistry 1961 The author via] Mr. R. E. TOIbGI“ oiiance into the c Trish to thank Dr. :oioreeeerch in t My intellectual Without the u: ethreat, much of 31': R. H. Schmidt . lgreet deal of or Et~irit in sharing I also wish t Eatlcnel Science 1 icing the Course ,f/ 1/ 1/ .. (‘- " V'sJ 7/(;./ AC KNOWLED GEMEN'I‘S The author wishes to acknowledge the technical help of Dr. N. E. Tolbert and, perhaps more important, his guidance into the climate of modern plant biochemistry. I wish to thank:Dr. R. C. Ball for the original stimulation to do research in this area and for his continuing interest in my intellectual development. Without the use of‘Dr. Harold Sell's laboratory equipment, much of the work would have been more difficult. Dr. R. B. Schmidt of Virginia Polytechnic Institute deserves a great deal of credit for his unselfish and scientific spirit in sharing research ideas. I also wish to acknowledge the support of the National Science Foundation and Michigan State University during the course of this research. 11 LIDGEURE R Polypho Special Separat or Phy: Some P acids mower 1c P'E‘IHODS Cultu: lsola Misce Preps EnZyl ESWES A as TABLE OF CONTENTS Page LITERATURE REVIEW Polyphosphates in Microorganisms with Special Reference to the Algae . . . . Separation of RNA into Chemically or Physiologically Distinct Fractions Some Properties of Isolated Ribonucleic BCIdSeeeeeeeeeeeeeeee INTRODUCTION . . . . . . . . . . . . . . . METHODS Culture . . . . . . . . . . . . . . . Isolations . . . . . . . . . . . . . Miscellaneous Chemical Determinations Preparation and Use of Charcoal . . . Enzymes . . . . . . . . . . . . . . . RESULTS AND DISCUSSION Anabaena Distribution of Phosphorus . . . Incorporation of P32-Phosphate . Location of Polyphosphate in theCell............ Characteristics of the Bacterium Isolated from the Culture . . . Characteristics of RNA-Poly- phosphate Phenol Preparations Changes Occurring as Cultures Reach Higher Densities . . . . . 111 1 3h 36 39 ha 57 so 65 65 67 67 70 7O 71 73 Chang with Dons Furt: Frac Stab Attev Arti. phos: Chlorella Chan Duri Chan Area Some 1301 RNA the Eff Cha Hype Pol} Enz: SWMY Changes in RNA-Polyphosphate with.Increasing Culture Density e e e e e e e e e e e 0 Further Characterization of the Fractionated RNA-Polyphosphate . Stability of RNA-Polyphosphates Attempts to Produce Synthetic or Artificial Complexes RNA-Poly- phosphate 0 e e e e e e e e e e Chlorella Changes in RNA-Polyphosphate During the Life Cycle . . . . . Changes Within RNA-Polyphosphate Areas During the Life Cycle . . Some Preperties of various Isolated Fractions of RNA'POIyphOSphate e e e e e e 0 Further Characterization of the Complexes 0 e e e e e e e 0 Effects of Adsorption on Charcoal e e e e e e e e e e e O Hyperchromicity of RNA- Polyphosphate . . . . . . . . . Enzymatic StUdiea e e e e e e e CONCLUDING REMARKS . . . . . . . . . . . 0 SUMMARY . O O O O O O O O O O 0 O O O C BIBLIOGRAPHY . . . . . . . . . . . . . . 0 iv 75 91 914 95 99 120 137 11414 1’47 15h 151: 166 169 172 .23 III team as v ‘O'OOW poms-00 Pr! TABLE I TABLE II TABLE III TABLE IV TABLE V TABLE VI TABLE VII TABLE VIII TABLE IX TABLE X LIST OF TABLES Changes in the orthophosphate content of the alkali-soluble fraction of Anabaena Distribution of phosphorus in Anabaeng harvested at two culture densitigs Parameters of typical phenol preparations of RNA-polyphosphate from Anabaena Nucleotide composition of the alkaline hydrolysate of an Anabaenm. RNA-polyphosphate phenol preparation Effects of mild alkaline hydrolysis on an RNA-polyphosphate phenol preparation and on synthetic poly- phosphate Changes induced by exhaustive dialysis against distilled water in the metachromasy and ultraviolet absorption of separated areas from the elution patterns in Figures 8-1h Effect of dialysis upon the ribose content of the eluates from a DEAE- cellulose column Concentration of ribose and total carbohydrates in a series of elution tubes from a DEAE-cellulose column Approximate base composition of a series of areas of RNA from a DEAR-cellulose column Effects of various treatments on the specific metachromasy of Anabaena RNA-polyphosphate Page 68 69 72 7h 7h 89 92 92 93 96 L XI 'L'E III 3.: XIII 'm‘ m a“ xv LEE XVI 1313 XVII Mn“ " ‘ XVIII 'TAELE.XI TABLE XII TABLE XIII TABLE XIV TABLE XV TABLE.XVI TABLE.XVII TABLE XVIII TABLEcXIX TABLEcXX TABLE XXI Effects of RNase on free polyphosphate, yeast-RNA, and RNA-polyphosphate Influence of light upon the ratio of total RNA 0. D. units to mgm. total phosphorus Base composition of the RNA in the pooled areas from the nine hour light stage of Chlorella Concentration of ribose in the pooled areas from Figure 30 (random Chlorella) Effects of various treatments on the metachromasy of area E RNA-poly- phosphate from random Chlorella (Fig. 30) Effects of charcoal and dialysis upon dialyzed and undialyzed areas B and D of Chlorella RNA-polyphosphate The effects of charcoal upon synthetic polyphosphate in a one percent NaCl solution ‘ The hyperchromic effects of boiling Chlorella RNA-polyphosphate for ten minutEs, followed by rapid cooling Release of orthophosphate by the action of the yeast polyphosphatase complex on known substrates Effects of yeast polyphosphatase on the metachromasy of synthetic poly- phosphate Effects of RNase and snake venom phosphodiesterase on the metachromasy of Chlorella RNA-polyphosphate vi Page 97 122 th th 1h8 155 156 158 159 161 163 ?hml EmeZ Inn 3 that isms a, Red: inocult Partial culture Schena1 culture Diagrar or the apparat Synchrc pyrenoi h“— Fractic Mans Change: POpula1 L. «sires 8'114 slut; 31in 15 RNA an. mlltur Total Ana denEIE @1333 16.27 Elu "the 28 ‘1 :‘E‘ll‘e 29 LIST OF FIGURES Page Figure l a, Medium.carboy; b, Anabaena inoculator h3 Figure 2 Partial diagram of the Anabaena culture apparatus hh Figure 3 Schematic diagram of the Anabaena culture system h6 Figure h Diagram of the plexiglass chamber of the mass, synchronized culture apparatus 52 Figure 5 Synchronization cycle of Chlorella pyrenoidosa (Van Niel 211} at 25° C. 56 Figure 6 Fractionation of Phosphorus components of Anabaena 58 Figure 7 Changes occurring in a typical Anabaena population during growth 77 Figures 8-lh Elution patterns of acid-insoluble RNA and phosphorus frochnabaena cultures of various densities 80-86 Figure 15 Total acid-insoluble RNA and phosphorus in Anabaena cultures of different densities 88 Figures 16-27 Elution patterns of total RNA and phosphorus from synchronized Chlorella cultures at various times in the life cycle 101-112 Figure 28 Total RNA and total phosphorus per 10 ml. p.c.v. of Chlorella during synchronous growth 11h Figure 29 Elution pattern of total RNA and phosphorus from a random culture of Chlorella grown under continuous high IIght intensity 117 vii lye” Elutior phosphc culture light 1 igres 31-314 Char e ync hrc fias535-38 Char Firm 39 Erato during Specifi RNA-pol dialysf Hyperwr polyphc against 319363 We Loss againsi F5335 Ill-Ah Loss 3m U firehfi dialys: RNA eli molecui Chloref Elutioz phospht after Infrar light Change 10 hou Change dialys ature bUffer Figure 30 Elution pattern of total RNA and phosphorus from a random Chlorella culture grown under continuous—16w light intensity Figures 31-3h Changes in Chlorella RNA during synchronous growth Figures 35-38 Changes in Chlorella polyphosphate Figure 39 Figure ho during synchrdfiaus growth Specific metachromasy of Chlorella RNA-polyphosphate complexes after dialysis against distilled water Hypermetachromasity of Chlorella RNA- polyphosphate complexes upon dialysis against distilled water. Figures h1-h2‘ Loss of Chlorella RNA upon dialysis against distIIIed water Figures hB-hh Loss of Chlorella polyphosphate upon Figure hS Figure M6 Figure A? Figure h8 Figure h9 dialysis against distilled water RNA elution curve of dialyzed low molecular weight complexes from Chlorella ’ Elution pattern of total RNA and phosphorus of Chlorella area E complex after dialysis Infrared spectrum of area D, nine hour light stage, synchronized Chlorella Changes in RNA-polyphosphate upon 10 hours dialysis at room temperature Changes in RNA-polyphosphate upon dialysis for 16 hours at room temper- ature against one liter of 0.01 M buffer, pH 8.0 viii Page 119 123-126 128-131 132 13h 135-136 138-139 lhl 1h2 lh6 150 152 P0] In the c1 :11 be dividec :hzsphoprote 1: rich are nail Fizsphates. '. Eat-fold alt 1‘. has been e Peale play th It is p0 Ether. The t LITERATURE REVIEW Polyphosphates in Microorganisms with Special Reference to the Algae Igportance‘g£_Polyphosphates i3 Algae In the cell of a microorganism the phosphorus contents can be divided into six main pools; namely nucleic acids, phosphoproteins, phospholipids, small molecular weight esters ‘which are mainly sugar phosphates, orthophosphate, and poly- phosphates. The amount of phosphorus in the cell can undergo many-fold alterations. In the process of these alterations it has been established that the ortho- and polyphosphate pools play the principle role. It is possible to separate these groos pools from each other. The bulk of the phospholipids can be extracted with a solvent such as ethanol-ether (3:1). It is also possible to extract, quantitatively, the ortho- and ptrlyphosphates along with some organic phosphates by using such solvents as hot five percent trichloroacetic acid or 0.1 N NaOH. Heating polyphosphates at 100° in l N HCl for seven minutes will completely hydrolyze these compounds to orthophosphate, which then can be determined accurately and selectively. This hydrolysis is mild enough that only minor amounts of organic phosphates are hydrolyzed. 1 in applicatic 379 tests has has :33). These 8185 me and the phOSE iifferent stages C aspborus ranged agaric weight. 'I ;:osphorus pools I 3.5 percent, and 1 at rest. By a d: 53 111311 pelymers as in the high p< Ehte Compounds 01 East 18 percent « Piliihosphate. I1 3115: over half 0: riii-LIPhosllhats. Tl aimmsive nat' worm and inpo \ d % In Order to .elildep a polyrh 0Ptho ‘ 2 An application of these techniques coupled with qualita- tive tests has been made in a study of Chlorella ellipsoidea (105). These algae were raised in a synchronous, mass cul- ture and the phosphorus in various pools was determined at different stages of the life cycle. The total cellular phosphorus ranged from about 1.5 to 2.2 percent of the dry organic weight. The amount of phosphorus in the organic phosphorus pools remained fairly constant at about 0.h to 0.5 percent, and the ortho- and polyphosphates accounted for the rest. By a division of the inorganic phosphates into low and high polymers it was shown that most of the polyphosphate was in the high polymer fraction. In a study of the phos- phate compounds of Euglena gracilis, Albaum (h) found at least 18 percent of the total phosphorus to be acid-soluble polyphosphate. In the case of Acetabularia, Schweiger (122) found over half of the total phosphorus in the acid-soluble polyphosphate. These studies and many others of a less comprehensive nature have established that polyphosphates are a normal and important part of the healthy algal cell. Definition In order to clarify the term polyphosphate, we shall consider a polyphosphate as a linear polymer of inorganic orthophosphate. There is some confusion in the literature, especially the older literature, due to the former use of the term metaphosphate. The latter term is now reserved for cyclic polymers of orthophosphate. A thorough up-to-date classification of poly- and metaphosphates can be found in itanheiner (99). :9 occur in microor ration of yeasts Lament form. it the present fictions about the tewhich seems val size polyphosphates {and at present. Among the algz Mg (125); 233 We: Siam ARM); and P teenreported in t Eater's yeast; (ll-t3 W (81), the bacteria p01yp % mo, in ’1 “a W1 (131), 3 Mattenheimer (99). Both poly- and metaphosphates are known to occur in microorganisms (73). However, with the possible exception of yeasts, the polyphosphates appear to be the important form. Occurrence At the present time it would seem that very few general- izations about the occurrence of polyphosphates are possible. One which seems valid is that most nonvascular plants synthe- size polyphosphates. No exceptions to this rule have been found at present. Among the algae polyphosphates have been reported in C_hlore11a (125); Euglena (a); Phormidium (31:); Eggs. @smarium, S iro ra, Mougeotia, Z ema, Navicula, Fragillaria, laucheria, Ulothrix, Oedoganium, Oscillatoria (66);: Acetabu- lagig (129); and Pseudoanabaena (5h). Polyphosphates have been reported in the following fungi: brewer's yeast and baker 's yeast (1)43), Agpergillus (97), Neurospora (51), finicillium (81), Merulius (1414), and Agaricus (82). Among the bacteria polyphosphates have been reported in Carme- Rgcterium (1h), Azotobacter (37), Mycobacteria (11:8), flcstridium (131), and Aerobacter (lhh). In the animal kingdom polyphosphates have been re- Ported in the fat bodies of the butterfly Deilephila (53), and the excreta of the wax moth Galleria (103). Polyphosphatase enzymes are known to occur in such vascular plants as rice (153), barley (113), and peas (106). Etiever, Keck l rshvescular l W' Eb gasphates in . Liter, beef mu state polypho atesult of ot a: cheracteri 37. the f 01101. 35°: another hrthere‘s re , live been sho 15‘s, it Seen Wise compour a; b" fOund 103+ wins P°leho EtacEIPOmat 1 35° 1°“ an nhOSphat 1; However, Keck (66) failed to demonstrate polyphosphates in such vascular plants as Pinus, Polypodium, Osmunda, and Psilotium. Ebel (314) also failed to demonstrate poly- phosphates in Escheréhia 9.3-2! Bacillus anthracis, sheep liver, beef muscle, and calf thymus. This failure to demon- strate polyphosphates must be qualified and reconsidered as a result of other investigations. An enzyme has been isolated and characterized from Escherischia 391.2; (71) which carries out the following reaction _i_r_1_ 1.1.33.2: x ATP-I-(PO‘3')n‘;=’X ADP + (Po-j)“; Also, another enzyme from g. 233}; (76) catalyzes the reaction: dG’I'P;=—-‘ P-P-P + dGR Furthermore, cell-free extracts of beef skeletal muscle (85) have been shown to carry out the reaction: ADP+ P-P==AMP + P-P-P Thus, it seems reasonable to doubt the complete absence of these compounds in _E_2. coli and beef muscle, even though they may be found only in small amounts. L_oc ation in Cell Polyphosphates have been detected in the cell by the metachromatic reaction, which is reviewed in a subsequent Section. This procedure doesn't necessarily detect the RNA- Polyphosphates which will be discussed in this thesis. In the marine alga Acetabularia the polyphosphate was found in spheres in the cytOplasm (129). These spheres Varied from 0.111 to 340011 in diameter depending upon the Physiological condition of the algae. They were concentrated . | . ‘50..d.‘."‘-"‘< "" ‘ ache tip of “I are even found aasive studY: I :fgreen 31586, the chlorOpl: 'fspyrenoids we date granules. fan, accumlate dam of unfert use polyphosph: 12" poly-phospha' lscillatoria ha: Serbst (94) Pep 131313 93233fi£ 9°1YPhOSphates , “Pies (79), . Eat in the par-1 tisnghip “Wee 513*. . .Val ants . Several 8’: 5 in the tip of the filament and the tips of the rhizoids and were even found in the gametes and zygotes. In a more compre- tmnsive study, Keck (66) found that in members of the family of green algae, Zygnemataceae, the polyphosphate was found in the chlorOplasts along with a high concentration of RNA. The pyrenoids were also surrounded by a sphere of polyphos- phate granules. He also found that Vaucheria, a coenocytic form, accumulated polyphosphate in small bodies in the cyto- plasm of unfertilized oogonia. Some diatoms were found to have polyphosphate granules in the cyt0plasm and Oedogonium had polyphosphate dissolved in the cytOplasmic vacuole. _Q§cillatoria had polyphosphate dissolved in the pseudovacuole. IHerbst (Sh) reports cytochemical evidence of nuclear equiva— lents in Oscillatoria and Pseudoanabaena which contain both jpolyphosphates, RNA, and DNA. In contrast are the findings of Krieg (79), who illustrated nuclear equivalents in Nostoc and Oscillatoria and showed metachromatic granula to be pres- ent in the periplasm. Krieg could show no cytological rela- tionship between these metachromatic granule and the nuclear equivalents. Several studies of Eggynebacterium.diphtheriae (101, 116) have shown the presence of polyphosphate-containing granules which varied in size with physiological conditions and seemed to be localized around a bacterial equivalent of nutochondria. In a study of living and fixed Nicobacterium thamnopheos (70) it has been shown that the polyphosphate was localized :granules ‘ 3531“: am sexed to 11! a: under-go I Us :15 L" 2:211:91 evi: inanimate, Ih‘ Fish basic p linked to RI; dilation H in (9 35:5 ASDQPQ‘ N 19 percent, t fined by 5 p “3) distir tile 11;) Cr C Weight, Was :53 up of t £1;th bESes Eating a SC 01 E 1:803 1 6 in granules which divide in the living cell, were feulgen positive, and gave a Millon test. These granules were ob- served to line up at the plane of division in dividing cells and undergo division. They were also shown to contain RNA. Using Aerobacter aerogenes, Widra (lhu) obtained cyto- chemical evidence that the volutin granules contained poly- phosphate, RNA, and lipids. These granules were also masked with basic protein. He concluded that the polyphosphate was linked to RNA and the RNA was linked to protein. Isolation Mann (9?) in l9hh, working with homogenates of the :mold Aspergillus, extracted polyphosphates with ice-cold 10 percent trichloroacetic acid (TCA) for 15 minutes fol- lowed by 5 percent TCA for 12 hours at 0°. In 19h9 Wiame (1&3) distinguished yeast polyphosphates of two types. One, made up of orthOphosphate and polymers of low molecular weight, was soluble in cold 10 percent TCA; and the other, made up of higher polymers, was soluble only in hot acid or dilute bases at room temperature. Lohmann (91) found that heating a solution of polyphosphates and nucleic acids in 0.1 N NaOH for five minutes at 100° would hydrolyze the glyco- Sidic bond of the nucleic acid, but would not hydrolyze the POlmphosphate. Lohmann also found that he could extract all c>f'the polyphosphates of brewer's yeast with boiling water, ENt it was necessary to use 2.5 percent TCA in the case of baker's yeast. Another technique deveIOped by Lohmann was the extraction of all polyphosphates with 0.1 M salt solution eisubsequent e; insphemolybdate zeta! may be us. Lies and La Balation for ye eluble fraction :czcentreted sal lie Chain-length 21 Uith an aver a 2135 fraction 8 536 to 0.05 N 1" Once the pc mated with e Witetee p0] 353092 at pH ; mm“ due t: filth ”1 °Que1 v M1 ted th 3333' The res eent ph°39hop us stem“ with a M adhated Elisa s°dium ac :3 Ecetato. Tr 7 and subsequent extraction of the orthophosphate as ammonium phosphomolybdate with isobutyl alcohol. NaCth, NaNO3, NaNCS, or NaI may be used. Lisa and Langen (88) have developed the most refined isolation for yeast polyphosphates. They obtained an acid- soluble fraction with a chain-length of three to eight, a concentrated salt and acid pH soluble fraction with an aver- age chain-length of 18-22, a fraction soluble at 00 and pH 10 with an average chain-length of 50-60, and an organically bound fraction soluble at room temperature on lengthy expo- sure to 0.05 N NaOH. Once the polyphosphates are extracted they may be pre- cipitated with any of a number of reagents. (A) Mann (9?) Precipitated polyphosphates with Pb(N03)2 at pH 1.8, and with 3&(N03)2 at pH 2, but this procedure lost most of the poly- phosphate due to hydrolysis. (B) He also treated an extract with an equal volume of ethanol, discarded the precipitate and adjusted the supernatant to pH 5.5 with either NaOH or NHhOH. The resultant precipitate contained about 13-16 per- c=ent phosphorus. By repeated reprecipitations he obtained a material with a phosphorus content of 23 percent. (C) Wiame (1‘43) adjusted a TCA extract to pH 14.5 with NaOH and then added sodium acetate until the solution was 1 M with respect to acetate. This was followed by the addition of Ba(N03)2 which precipitated a good yield of polyphosphate. (D) Inor- Sanic phosphates including polyphosphates have been precipi- tated by various monoamines, diamines, hydrazines, dibasic gino ac ids, :recipitation :clyghosphate agitation vs are first mi reszlting pre grezipitated elcebol (91). Applica‘ has resulted ’I l .30) of a 1‘ that in the . 3f P013'ph08p length or te :tate accoun 3511. 4-3 “1"" \551‘ poly:- 8 amino acids, dipeptides, and guanidine compounds (35). This precipitation by amines was selective for highly polymerized polyphosphate at pH's of 2.0 to h.0. Completeness of pre- cipitation varied from 50 to 95 percent. If the extract tare first mixed with an equal volume of ethanol and the resulting precipitate removed, the polyphosphate could be precipitated from the supernatant by adding Mg012 and alcohol (91). Application of various combinations of these methods (has resulted in phosphorus fractionation data for Acetabularia (136) of a fairly comprehensive nature. These data indicate 'thmt in the case of this alga there are no detectable levels of’polyphosphates between orthophosphate and polymers of a length of ten phosphates. They also indicate that polyphos- phate accounts for the bulk of the total phosphorus of the cell. Physical Properties At present no one has isolated polyphosphate from an algal source and determined such properties as molecular weight. A water soluble, non-dialyzable polyphosphate has been isolated from Aspergillus giggg and an average molecular Weight of 6000-7000 obtained (59). Similarly two types of P01yphosphate were characterized from yeast. One had an average chain length of about ten phosphates and the other had an average length of about 50 phosphates (155). The larger polymer was found in greater amounts. Lisa and Langen 55}, as discusse sphosphate in :rgaficelly boun< :1 this fraction :ates. Electro iziicated that 3 One study k spolyphOSphm Yeast po'. Graham's Plrophos ”“1 these data {53 as mIllivale I. Lr. It has be‘ Lmersibly b] {asphates in iialyzability was tested. I slightly taste ‘ .H .. at“ ts Salt, lite fairly rs n a Stu. 5-; me 1 9 (88), as discussed earlier, characterized four types of polyphosphate in brewer's yeast. In a further study of the organically bound fraction (89) they showed that 75 percent of this fraction had an average chain length of 165 phos- phates. Electrophoretic behavior of this polyphosphate indicated that it was composed of a variety of chain lengths. One study has been done on the heat of hydrolysis of the polyphosphate from yeast (15b). The results were: Yeast polyphosphate A H =3 10 Kcal./anhydride bond Graham's salt A H I ll Kca1./anhydride bond Perphosphate AH= 9 Kca1./anhydride bond From these data we classify the polyphosphate anhydride link- Age as equivalent to the "high energy" anhydride linkage in ATP. It has been shown that low-frequency ultrasonic waves irreversibly break the phOSphate anhydride bonds of poly- Phosphates in aqueous solutions (23). In the same study the dialyzability of various polyphosphates and metaphosphates was tested. It was found that the metaphosphates dialyzed Slightly faster than the corresponding polyphosphates. Graham's salt (Wt. aver. mol. wt.= 15145) was found to dia- lyzo fairly rapidly and a series of other polymers with weight average molecular weights of up to 17,580 showed gradually decreasing rates of dialysis. In a study of long-chain polyphosphates Malmgren (96), made use of the electron microscope and streaming birefring- ence in addition to ultracentrifugation and diffusion rates. Econcluded that as, that th" new of the raiser a Winkle. :L’frection that fling-chain PC trims ways 5‘“ “distance 0' teoistance 0 In a stud: :elyphosphete 3: electrolytes . salt solutions zen in conoen 7.1“» "' be: a a a 10 ER concluded that the shape of the molecule changes with the nmdiwm, that the molecules are largely solvated, and that 'um shape of the molecule is not a perfect sphere, but ruther a wrinkled chain. Thilo (137) demonstrated by X-ray diffraction that the phosphate tetrahedra of various salts of’long-chain polyphosphates are wrinkled and spiraled in various ways such that the sodium salt has three phosphates in a.distance of 7.3 A and the silver salt has four phosphates in a distance of 6.1 A. In a study of diffusion rates of synthetic and yeast polyphosphates, Katchman (65) found a marked effect of electrolytes. Diffusion was more rapid~in water than in salt solutions and the rate was also more rapid in dilute than in concentrated solutions of polyphosphate. Methods of Analysis The analysis of polyphOSphates is a difficult problem 'Hhich has not been completely solved to date. The qualita- tive analysis may be divided into chromatographic, electro- Phoretic, colorimetric, and spectroscopic methods. It has been known for some time that very dilute solu- tions of polyphosphates give a metachromatic reaction with the basophilic dye, toluidine blue (1&2). With a given poly- mer a maximum color production takes place at a certain ratio of phosphate to dye and color production declines on either side of this ratio. This'color develOpment is inhib- ited by salts. The metachromatic reaction is also given by icrosco {uh 0.1 1:15 10 a: ‘oe s T2636 re Fiéapha- : 101‘) 11 sulfuric esters of polysaccharids, by nucleic acids, and by polyanions in general (66). A weak reaction is given by ATP (13). Bradley (12) has made a study of the chromatic reactions of a variety of dyes with several polyanions. If cells are dyed with toluidine blue and fixed on ndcroscope slides, the nucleic acid color can be extracted with 0.1 N HCl and the polyphosphates can be extracted with cold 10 percent TCA. Any remaining colored cell material can be assumed to be sulfuric esters of polysaccharides. These reactions can be used to qualitatively identify poly- phosphates in cells or extracts. It has been shown that methyl green and pyronine give almost identical cytological reactions (36). One attempt has been made to utilize the metachromatic reaction as a quantitative method by control- ling temperature, pH, salt concentration, and time of re- action (28). This method was still only semi-quantitative due to interference from other biologically occurring mate- rials and the variation in color development with the length Of the phosphate polymer. This variation was thought to be Gum to the interference of the doubly-charged and phosphates (135). One-dimensional paper chromatographic procedures for Polyphosphates have been developed (9, 33, h9, 137), and two-- dimensional paper chromatograms have been used to separate nIstaphosphates from.polyphosphates (36). Attempts to elute Spots and analyze them by micromethods have only succeeded in giving semiquantitative results, and there is some doubt zezive so. :se of Do {sates an is limit pzsptate The teen dete iet'u'een 8 Elite Was i‘eaker ba Upon 550.280 m “wt 350 intensity 9 0‘ 901mg 12 if they are any improvement over the measurement of the density of spots after spraying with a color-developing reagent. Continuous paper electrophoresis has also been developed and under ideal conditions it can yield a quanti- tative separation of various phosphate polymers (117). The use of Dowex-l ion-exchange resin to adsorb the polyphos- ‘phates and dilute HCl-KCl mixtures to elute the components luas limited usefulness for the separation of the oligopoly- phosphates (72). The infrared spectra of several polyphosphates have been determined (26). Orthophosphate had a broad absorption between 8.7 and 10.0 u, and a linear, long-chain polyphos- phate was found to have a strong band at 7.7 to 7.9uand a weaker band at 11.5 to 11.8 11. Upon eXposure to ultraviolet light in the region of 260-280 mu or 296-313 mu, polyphosphates fluoresced at about 350-360 mu rather markedly, and a tendency for the ilntensity of the fluorescence to be dependent upon the degree 01' polymerization has been noted (2). The state of quantitative analysis of polyphosphates is not very well advanced. Of the many methods of analysis for total phosphorus, that of King (68) applies well to this t"3pc of material. Orthophosphate may be determined quite atBcurately (92). Furthermore if the only inorganic phos- Phates present were orthOphosphate and pyrophosphate, these compounds could be accurately determined (39). Beyond this simple beginning a certain amount of guess work enters the picture. I .ja'ltitativ 1) 331 for nails to 27.8 . LO h." tomato I 13 picture. It is known that all inorganic polyphosphates are quantitatively hydrolyzed to orthophosphate by boiling in 1 N H01 for seven minutes. Most organic phosphates are stabile to these conditions, but this is not universally true. Lohmann (91) determined orthophosphate (0:), ortho- phosphate after seven minutes hydrolysis at 100° in l N HCl (5:). and orthOphosphate after 30 minutes hydrolysis at 1000 in 1 N HCl (Av). He then postulated on the basis of some experimental work that (A1) - (3:) as polyphosphate. Ecolggical Considerations The existence of polyphosphates in natural populations of algae and the quantitative importance of this type of phosphate has not been directly demonstrated. However, in view of the following literature reports, such an occurrence 01‘ polyphosphate should not be in serious doubt. Lund (9b) in his studies on the annual spring blooms of Asterionella formosa in England found the blooms to be almost entirely Composed of this algae. In this case available orthophosphate "as usually in the range of one to four parts per billion (P.p.b.) and silicon was shown to be the limiting factor in growth. It was found that the total phosphorus per cell was InItch higher at or near the maximum period of the bloom. K6‘tchum (67) in studies of a pure culture of Chlorella has shown that under ordinary culture conditions the phosphorus 1"0 nitrogen ratio was about one to three, but under conditions Of deficient phosphate the ratio became about one to 17. ‘ l ' ‘ ‘fi-—_e_ . 5'2) veriati< gages in t] It is i: favcra'fle to eiefor suc Ezseve (58) lsaemaoii the) dove 5);,p,b. istors as t Ester contai (1:0)) is not EPeitide ( f The ab: 75°Sphate 1) ”in ““93 Ma 15p «‘22‘ . ‘3 (<2). AlEae 11: Such variations in total phosphorus are probably due to changes in the polyphosphate pool. It has also been found that Chlorella accumulates polyphosphate in the absence of 602 (150). It is interesting to contrast the type of environment favorable to the growth of blue-green algae with that favor- able for such algae as the Asterionella discussed above. Guseva (58) found that under natural conditions Anabaena lemmermanii required 870 p.p.b. of phosphate-phosphorus for optimal development and Aphanizomenon flos-aquae required 260 p.p.b. These values may be affected drastically by such factors as the nitrogen source. There is evidence that lake water contains a factor which aids in the uptake of phosphate which is not present in synthetic media. This factor may be a peptide (58). The absorption of orthophosphate and formation of poly- Phosphate in yeast can be carried out anaerobically but re- cluires potassium ions (119, 1143). This potassium require- m°nt is due to its effect on the yeast cell wall permeability (153). In the case of diatoms, Fe(0H)3 is believed to play a role in the uptake of orthophosphate. Fe(OH)3 forms a c’Olloid which adsorbs phosphate and is, in turn, attracted to the cell membrane of the diatom (52). It has been sug- gested that algae precipitated to the bottom of the sea were the source of the present-day sedimentary phosphate rocks (22). Algae can be controlled in water for cooling or swimming " nsw 3 ‘n ‘. tezmete Iiey were timers miners :eztret: :hates c § 8 Mom L W ‘_U‘_.'—-.—— 15 pools by inhibiting their growth with the heavy-metal salts of polyphosphate (112). Gross Physiological Effects 93 Higher Animals Labeled polyphosphates, when injected intravenously into dogs, were stored in the liver and spleen. They were then degraded to orthophosphate ((47). When trimetaphosphate and tetrametaphosphate were injected intravenously into rabbits, they were excreted intact in the urine (145). The linear polymers were hydrolyzed to orthophosphate. All phosphate polymers were hydrolyzed when taken orally. When high con- centrations were injected intravenously into rats,polyphos- phates caused severe acidosis, the formation of a calcium complex, and cardiac arrest U46). It has also been shown that both pyro- and tripoly- phosphate significantly inhibited oxidative phosphorylation in isolated mitochondria (138). Phosphate glass failed to have an effect . Biosynthesis In order for an organism to carry out the biosynthesis of polyphosphates it must generate "high-energy" phosphate carriers such as ATP. The formation of this carrier may be aficomplished by photosynthetic phosphorylation or respira- tory oxidative phosphorylation. In the photosynthetic bacteria which do not have their Brena enclosed in a chloroplast, 002 fixation takes place in the cytoplasm (142). This may also be the case in blue-green __-—_—.._. 'w. ‘12: p32 l6 algae. The ATP which is produced by photosynthetic phos- phorylation is very rapidly utilized by two competitive mechanisms: C02 fixation and polyphosphate formation. Hassink (1)40) found evidence for the accumulation of a phos- phate compound in Chlorella other than ATP after two minutes exposure to light. Wintermans (151) found the uptake of orthOphosphate by Chlorella to be rapid in the light and more rapid in the absence of 002. He concluded that there was a conversion of photosynthetic energy to stored polyphosphate. He supported this conclusion by extracting polyphosphate from the cultures. Early workers assumed that the mode of synthesis of polyphosphate chains was the addition of phosphate units one at a time to a short starter or to orthOphosphate itself. In 19h9, Wiame (1143) postulated that two systems might exist, one for the biosynthes is of the longer polymers and one for the formation of the shorter ones. This came as a result of his studies of the acid-soluble and acid-insoluble polyphos- phate pools in yeast which had been exposed to various changes in their environment. Unfortunately no enzymatic studies have been carried Out on polyphosphate formation in photosynthetic organisms. However, some studies have been done on yeast and bacteria. In 195).; Hoffman-Ostenoff (55) reported the finding of an el’lzyme in yeast which carried out the following reaction W1th P32-labeled polyphosphate: ADP-l- (1114192035? ATP32+ (NaP3203)n_1 fge ottira section A 11?. In. :..£ 9 . 5..“ 11.0! :farele L'. .. a .45 3.11)“). tgnesi‘m 17 The optimum pH was found to be 6.7, but the reversal of the reaction was not demonstrated due to a lack of P32-labeled ATP. In 1955 Yoshida (155) mentioned "metaphosphate synthe- sized from orthOphosphate or ATP by the enzyme juice ex- tracted from yeast" and referred to a previous obscure paper (152). Finally in 1956 Kornberg (71) reported the isolation of a relatively stabile enzyme from Escherischia 9.215; for the synthesis of polyphosphate. This enzyme required magnesium ion ()4 x 10"3 ); was unaffected by potassium, arsenate, or dinitrOphenol; had an optimum pH of 7.2; was completely inhibited by fluoride; and carried out the re- action: x ATP + (Poppa-‘1: ADP + (P03)n+x primer In a later paper (75) Kornberg also illustrated the reversal 01‘ this reaction and showed that CDP, UDP, GDP, and AMP had little or no reactivity in this system. A very interesting f2Lnding was that low concentrations of ADP inhibited the 1Tormation of polyphosphate in this system. This fact places , considerable doubt on the role of such an enzyme system in the biosynthesis of polyphosphate. Kornberg accepted the Possibility that two systems are in operation for the syn- thesis of low and high polymers as discussed above. Kornberg's 1‘1na1 product was quite large, since it was quantitatively Precipitated by albumin at pH h.0. Kornberg (72) suggested a mechanism for the formation Of tripolyphosphate: aka-"I ‘ A} 0»! in en ten isola :filize UT Emma measured, Tisen yr Tsfious sj firection-s “is to f It he TRCVep ( )1 130). 3’75P101 lea “e 97017 ‘ ,Ms waders: l8 aden late P-P + ADP king” 4 P-P-P + AMP Eh also suggested that the large amounts of pyrophosphate formed by pyrophosphorylase reactions might undergo reactions such as: P-P+ (P03)n= P1+ (PO-3')n+1 An enzyme system similar to that of Kornberg's has now ‘been isolated from Azotobacter by Zaetseva (157). It can not txtilize UTP or ITP in the forward direction or IDP, GDP, or IDDP in the reverse direction. Since total polyphosphate was zmeasured, we can be sure that net synthesis was demonstrated. Thais enzyme was tested for activity on polyphosphates of vsudous sizes and only showed activity in both reaction ciirections with those of high molecular weight. ADP was found to inhibit the forward direction of the reaction. It has been thoroughly demonstrated that the metabolic ‘turnover of all types of polyphosphate is very rapid (7, 80, 8h, 130). By adding P32-1abeled orthophosphate to the media ifor’various lengths of time and at various stages of the ilife cycle, and by intricate polyphosphate fractionation IIPocedures; two groups of investigators working with dif- I'orent organisms have arrived at the same conclusions (80, 814, 156). One conclusion was that the largest polymers 91‘ polyphosphate were bound rather tightly to an organic compound. Another conclusion was that, on the basis of both 8Pacific activity and total activity, this type of poly- a; titall As a zeterel wc hes. thee i5, 20, 3] issue deg E? or m Two 1 l9 phosphate was the first to become P32-labeled and there- after the other lower-weight polymers became labeled in descending order until the classical ac id-soluble fraction was finally labeled. As a result of studies of RNA-polyphosphate complexes, several workers have postulated that polyphosphates might be synthesized on the surface of RNA or a nucleOprotein (6, 20, 31). These long polymers could then separate and become degraded either by means of tranSphosphorylation to AEDP or AMP or by means of polyphosphatases. Two reports in the literature tend to support a conflict- iJlg view that very low molecular weight polymers of phosphate rmight be made directly from phosphate in some cases. Szulmajster and Gardiner (131) using a homogenate of a luutant of Clostridium found that 37.5 percent of the P32-labeled orthophosphate was incorporated as pyrophosphate, 2.3 percent as tripolyphosphate, and 22 percent as unidenti- lfied.polyphosphate. This mutant uses creatinine as its major energy and nitrogen source and 002 as a major carbon source. A possible reason for these results could be a very actdve polyphosphatase system.and 1 rather severe extraction Procedures which might have broken down the higher molecular "9 ight polyphosphates to pyro- and tripolyphosphate before 1alley were identified. More work is needed to elucidate this point. In the second case Goksoyr (uh) found that the basidio- mycete fungus Merulius lacrymans, upon exposure to P32-1abeled v-- , - m. 5w»: "‘ ‘ ' gsphEtO 3 first and ‘ ta: ATP a insight 1‘3 and LD Sever 53m to p 715) four. gate cont seat of th 3:22;: Wind iiiition c sites. 1 -. A *3 etfeot. 20 phosphate for various time intervals, labeled pyrophosphate first and the perphosphate had a higher specific activity than ATP at 30 minutes. It was also found that high molecu- lar weight polyphosphate was not labeled rapidly while both GDP and UDP were labeled rapidly. Several conditions other than 002 deficiency have been shown to promote unusually high polyphosphate levels. Winder (1146) found that mcobacteria tripled their inorganic phos- phate content in five hours and underwent a tremendous enlarge- ment of their metachromatic granules when 2.5 percent tetra- hydrofurfuryl alcohol was added to their medium. In a later study Winder (1149) found lesser but similar effects upon the addition of ethanol, tetrahydrofuran, or butanol to their medium. Propylene glycol, butyrate, and chloromycetin had no effect. Wiame (11:3) studied the effects on the polyphosphate in yeast of alternately placing the yeast in phosphate-free and Phosphate-containing media. Upon exposure to phosphate, Phosphorus-deficient yeast, which had very little polyphos- Phate, formed large quantities of both low and high polymers or phosphate. Next the orthophosphate and high polymers were 1"educed. Then the low polymers were rapidly utilized and the yeast returned to their normal condition with regard to phosphorus pools. Thus, eXposure of phosphorus deficient °1‘ganisms to high phosphate concentrations could be a method °r improving isolation yields. It has been shown in cultures of Mnebacterium It . ' 4-—§-'—nv——t> ‘V Esteem It was to III-thesis Viz—hem; t ifiatoie. Q The ten Shox fiatase 1 :5 3.5011 . 1: i!.&.fpe 31033 th- 1| 21 ) diphtheriae (101, 115), that glucose in the medium depressed polyphosphate and increased RIC-A, but dinitrOphenol or malate had the opposite effect. There is some evidence that calcium might be involved in the biosynthesis of polyphosphate. It has been shown (37), ' that calcium was required for both nitrogen fixation and ’ polyphosphate synthesis in Azotobacter vinelancli. ) The phosphorus metabolism of Acetabularia mediterranea has been studied under normal and enucleated conditions (122). It was found that growth, polyphosphate synthesis, protein without the nucleus, but at a decreased rate. k AAA I I synthesis, and phosphorus metabolism in general proceeded I gatabolism The catabolism of polyphosphates in microorganisms has been shown to proceed by both energy conserving and phos- phatase mechanisms. The energy conserving mechanisms will be discussed first. It was found by Winder (1147) that in ) cell-free extracts of flycobacterium under anaerobic condi- ) tions the reaction: , (NaP03)n+ Glycerol iE-gF‘E-t glycerol phosphate-4- (NaPO3 n-l took place if polyphosphate, glycerol, and ATP or ADP were added. A further purification of this enzyme system was carried out (1149) and resulted in the characterization of the enzyme polyphosphate~AMP-phosphotransferase, which c atalyzed the re action: . h: I . . as 3L?" 1 null-Hr t: ._.V=U..a t. 3131 in If their The 3:25:01: 333,716 1. ~"' 4» 1 ‘* dune ’5: ~. W" onfl I "'35P. 4. ‘ "HS v] H w. ) 22 AMP-P (NaPO3)n-.=-f=‘ ADP + (NaP03)n_1 The enzyme was located in the 25,000 g supernatant fraction, was stimulated by 3 X 10mll M Mg‘; would not utilize pyro- phosphate, and had a pH Optimum of 6.3. If we consider the previously discussed enzymes which carry out the transphosphorylation to AMP and ADP and which I seem to have equilibria in the direction of ATP formation r (55, 71, 75, 152, 155, 157), they constitute a system to explain the breakdown of polyphosphates with conservation of their "high-energy" bonds as either ADP or ATP. The only report of an energy conserving enzyme for the w—_———_F—-——— metabolism of metaphosphates is that of Rafter (107). This enzyme was found in yeast and carried out the phosphorolysis of trimetaphosphate to tetrapolyphosphate. Since yeast is the only organism in which cyclic metaphosphates have been demonstrated as a major constituent, this was the most likely source for such an enzyme. This is the only case reported at this time of an enzyme which carries out the phosphorolysis of a cyclic compound. ) Another interesting enzymatic system, which has been reported by Rafter (108), utilized pyrophosphate and glucose to form glucose-6-phosphate and orthOphosphate. Such a system, if it is found to occur widely, could explain another method of utilizing the energy of the oligopolyphosphates as they are broken down. Thus far it has only been demon- strated in mouse mitochondria. ?4. -h m» a Wren- 41’ Poly- I wring. tied yeast :1: Exam a1 ripalyphom silt. Poly tibarley I ‘2: (97) f site of A_s Site h.2 12:5 stimul 5799?“ its Pi‘ificetio "~ a male Mop at 13316 pH 3 Famous 1 Epitaph; 32- increase ”More “15 inhibj Tie méh mc Emitive. Eoptina 1 Kmit: Hens ye' ——— _.__—...———- 23 Poly- and metaphosphatases have been found widely occurring. Mattenheimer (98) found frog muscle extracts and dried yeast to be capable of splitting all phosphate polymers, but human and rat liver could not split polyphosphates above tripolyphosphate and human kidney doesn't act upon Graham's salt. Poly- and metaphosphatases were found in rice (153) and barley (113). No work has been done with algae, but Mann (9?) found a polyphosphatase in both the cells and the media of Aspergillus 93:59:. He found the Optimum pH to be 3.7 to 14.2 Lindeberg (86) found that ferrous and cupric ions stimulated this enzyme's production, while high Zn” stopped its production in _A_. 92593:. Malmgren (96) studied the polyphosphatase complex of A. £13533 after a 20-fold purification. He applied this complex to a polyphosphate with a molecular weight of one million and the action seemed to stop at about the level of tetra- or pentapolyphosphate 1f the pH was controled and manganous ion was added. Manganous ion was an activator for the high molecular weight polyphosphatases but not the lower molecular weight ones. It increased activity 30-fold. Mg"! Cal"! and Zfi'tnay act as, activators also. Fluoride, phloretin phosphate, and citric acid inhibited the low molecular weight polyphosphatases. The high molecular weight polyphosphatases were more heat sensitive. Phosphatase activity was found to have different pH Optima for different polyphosphate substrates. Kunitz (83) has crystallized the pyrophosphatase from baker 's yeast. It is a protein of the albumin type with a elecular we :1 about pH azzivetor, e zegerature 2:7 active?» £51000 col In 195 L‘geest e7 trime‘ tripe pyroe 1 ##fi' 2h molecular weight of about 100,000, and an isoelectric point of about pH h.8. It requires Mg“; Mn”, or CO++ for an activator, and is inhibited by Catt 0ptimum.pH is 7.0, temperature 140°, substrate concentration 3-14 X 10’3MMauP207, and activator concentration 3 X 10'3 M Mg“. Maximum rate is 1000 moles of perphosphate per mole of enzyme per second. In 1956 Kornberg (7h) illustrated the following system in yeast extracts: trimeta- phosphatasf. tripolyphosphate tripoly- phosphatase pyrophosphate 35-55-1352; as e »2 orthophosphate trimetaphosphate tripolyphosphate —)pyro-P + ortho-P In the same year Mattenheimer (99) Published the results of a more extensive study. Working with brewer's yeast, pri- marily, he found the Optimum pH to be close to 7 for meta- phosphatases and close to 8 for polyphosphatases. In the case of perphosphatase he postulates the following mech- anism of action: E +MgPP._-==§ EMgPP==3E +Mg 4-2? ml! :er EPP s( MgPP) 2 By means of ammonium sulfate fractionations Mattenheimer obtained two fractions with specifically higher activity on pyrophosphate and tripolyphosphate respectively. Similar separations were made on alumina, calcium bentonite, and by means of electrOphoresis. By studying the products of P°1thosphatase activity on high molecular weight substrates, i:.‘¢“‘h {W‘ inc.- 6.“ tall. “la. poly- oligo a Ufihephosl in e: F‘Jified j ‘mtion «ea ‘239503 a the te 25 Mattenheimer was able to show the following scheme of break- down: poly-HE = 27-30) d°P°1¥m°rasis poly-P65 = 10-?) + J a little ortho-P depolymerase oligopoly-PGE .-.- 14-10) + a little ortho-P Eligopolyp hosphatases trgoly-P + orthO-P trimeta- phosphatase J trimeta-P — tetrapoly- phosphatase - tfifgmeta' - tetrameta P phosphatase #tetrapoly P From tripolyphosphate Mattenheimer's scheme proceeds in the same manner as that of Kornberg's. In this manner yeast are able to hydrolyze high molecular weight polyphosphates to orthophosphate . An enzyme from Mnebacterium xerosis (102) has been purified lOO-fold which is unusual in that it causes no formation of short polyphosphate polymers, but rather only orthophosphate. This suggests that it Operates specifically on the terminal phosphates of the chain. It is also unusual in that all metal ions tested, including Mg)": inhibited activity and EDTA stimulated activity. Another unusual enzyme or enzyme complex has been studied in peas (106). In this case no separation could be achieved between phosphomonoesterase, and polyphosphatase activity. The enzyme attacks trimetaphosphate, Graham's gizsgh te tall. In : 21359313131 5361‘ al 3'19! ' 3 ET“. in.‘ 0" Fl 26 salt, tripolyphosphate, nucleoside triphosphates, and fi-glyoerol phosphate but not tetrametaphosphate. It is inhibited by fluoride and molybdate, and is unaffected by Mg". One possible physiological role of polyphosphatases would be as a mechanism for the breakdown Of external poly- phosphates in order to make the phosphate available to the cell. In studying either biosynthesis or catabolism of poly- phosphates several facts should be kept in mind. Ca'H'and other alkaline earth ions are known to catalyze the non- enzymatic breakdown of high, but not of low molecular weight polyphosphates in alkaline solutions (89). It has also been shown (136) that there is a chemical equilibrium in neutral aqueous solutions at 60° as follows: trimeta-P + ortho-P= tetrapoly-P Similar interconvers ions between other polyphosphates and metaphosphates at elevated temperatures should be expected. Organic Gogglexes Under this general heading will be discussed a number of compounds and complexes which have been shown to contain polyphosphates or which may contain polyphosphates. In the presence of magnesium ion; pyrophosphate, tri- POlyphosphate, ATP, and Graham's salt all form dissociable cOI‘IPJ-exes with actomyosin and the actomyosin is split into myosin and actin (1). “flood-'3‘ a‘.a_‘- ?i3;hor 11:5 Eli; $5.2m} fitted . 3% be: {1635 t 27 The longer polyphosphates are coprecipitated with the polycations of proteins when TCA is added, but the short polymers are not (614). This accounts for the "insolubility" of the higher polyphosphates in cold TCA. From a physiological point of view, the most interest- ing of the polyphosphate complexes studied to date are the RNA-polyphosphate complexes. For many years a close cyto- logical connection has been noted in a wide variety of organisms between RNA and polyphosphate. One of the earliest direct approaches to the relationship was in 1953 (32). At that time it was reported that isolated RNA contained excess phosphorus and that this phosphorus was ac id labile. If this RNA was incubated with myokinase and ATP, the excess phosphorus could be increased. In 1956 Winder (1148) re- laorted the isolation from Nygobacteria of a fraction contain- ing both RNA and polyphosphate. In an attempt to separate these two components neither of the following was effective: 1. Extraction for 2).; hours at 19° with 0.01 M bicarbonate buffer, pH 9.0. 2. Extraction for 2h hours at 370 with saturated aqueous urea, followed by l M NaCl at 19°. About the same time Belozersky (6) reported finding that the acid-soluble polyphosphate of yeast combined with pentose polymers and RNA. In 1957 the same group (7) showed that the phosphorus entering into the cellular composition of A3 or illus niger was first found in the acid-insoluble P°1YPhosphates and that this fraction contained a large Co's. ‘J 331“» of J zezteined t3 tithe excl ;cl;r;':os;he feast-Fill t sittiagne: affected. iate for 3 3338.110 m use and cTimica] Efluticm 28 amount of RNA. After many reprecipitations the RNA still contained three times as much phosphorus as to be expected and the excess phosphorus was shown to be in the form of polyphosphate. This group also showed that mixtures of yeast-RNA and polyphosphate could be separated by treatment with magnesia mixture, but that the natural material was not affected. In 1958 Langen (8h) reported the same general type Of ciata for yeast, but he did not investigate the nature of the (organic material combined with the polyphosphate. In 1959 ILiss and Langen (88) reported that the release of this <>rganically bound high molecular weight polyphosphate into solution with 0.05 N NaOH followed first order kinetics and teas quite temperature dependent. He suggested that a chemi- <3al reaction was involved in the release. In 1960 the same 3gmeup (89) further characterized this fraction of organically loound polyphosphate. If the yeast were pretreated to remove IRNA either by the action of RNase or dilute alkali and salt ‘extraction, the kinetics of the polyphosphate extraction were ‘umaffected. When 75 percent of this polyphosphate fraction was carefully purified, its average chain length was 165 phosphate units, corresponding to a molecular weight of 19.500. The authors expressed the belief that there was no‘connection between RNA and polyphosphate on the basis that : 1. Most of the RNA can be removed by salt extraction without simultaneously extracting polyphosphate. "flee fee iii not 6 223 Le“ ~. .1?‘ s. .3 "’l a. 1.0". a "‘V' ~.L'.A ' - 55:!‘1‘L, I'VA y existed tie fr: fi— 29 2. The 75 percent Of the organically bound fraction of polyphosphate which can be extracted by dodecyl- sulfate can then be treated with charcoal, which removes the RNA and not the polyphosphate. These facts did not prove that an RNA-polyphosphate complex did not exist. They did indicate that the yeast which Lies and Langen studied probably contained a considerable amount of RNA and polyphosphate which was not complexed. In another case Schweiger (122) applied charcoal adsorp- 'tion in a fractionation of the phosphorus compounds of 3Aeetabularia. He found that varying percentages Of poly- ;ohosphates, both acid-soluble and acid-insoluble, were smdsorbed by the charcoal. In these cases there may have (existed a complex between the RNA, which was adsorbed, and the fraction of polyphosphate which was adsorbed. In 1958 Griunberg-Manago (D8) reported the isolation of an enzyme from yeast which synthesized RNA polymers. It teas not typical of this type of enzyme since exchange was 3inhibited by ng'and the enzyme was able to utilize GDP. IPolymer formation required Méfl'and was specific for nucleoside 111 enlarged i tip bacterial filter detachable polyethylene squeeze bottle Figure 1b, Anabaena inoculator (a momipet) air exhaust line bacterial filters I / ..J glass / “ exhaust tube 1 \’ / r’ A \ ,‘lJa 1....‘rvr—npl \ 1' support air inlets rack Figure 2 partial diagram of the Anabaena'culture apparatus glass wool plus drain ”.5 1.3.11: iii a ass-i 1 .L‘ H1 09V it"s}. “‘Vol 3 I J, 3‘ ‘3‘;‘ in I 9“"! dude 115 This allowed for adjustment in height until an intensity of about 500 footcandles was obtained. A compressed air line and a carbon dioxide cylinder with reduction valves were used for aeration of the algae cultures and circulation of the water bath (as explained later). For the culture flasks themselves two six-liter Florence flasks were used in an inverted position. This was the largest practical size since light rapidly became limit- ing in a dense culture. This type of flask was necessary because of the ecology of the organism which, without rapid turbulence, formed plate-like masses on any surface to which it was exposed. The air line bubbler was a one-liter Erlenmeyer flask with a four-hole stopper, Figure 3. This flask was main- tained about one-half full of distilled water and it both moistened the air stream and gave a method of adjusting the relative volumes of carbon dioxide and air. Carbon dioxide should never exceed one percent by volume. The water bath bubbler valve could be used as a fine adjustment of the amount of carbon dioxide flow. The portion of the apparatus enclosed within the block labeled "sterile zone" was sterilized as a unit. This was done as follows: 1. The two Florence flasks were put in an upright position and filled with medium. 2. The stoppers and everything else involved in the "sterile zone" were assembled and all clamps were Closed 0 1.1.6 .Eoumhu assuage «neurone one mo_ssnms«e carcasses .m easmHm fie 111111 oddfi.flfldfimmm. 11111 G 6“ i. u W0 & . \ 11%.. {I a a- a to FIIIIIIII... .IIIIIIIII... dogmas—coda no.“ 8.3 0.3m M395» More?" Hmoawnsm phone honfleo Eaves Son.“ and scan madame» hone?" Heoawasu MOOH moo on: has flea W nude songs has I“ (I. v.’ 'd 3. h. 5. M7 Clamp 3 was Opened. The whole "sterile zone" was autoclaved one hour and cooled to room temperature. Clamp 3 was closed and the Florence flasks were inverted and placed in Operating position. The bacterial filters were connected to the line from the air-line bubbler and clamp 3 was opened. After several hours of circulation the medium should be completely in solution. The Florence flasks were inoculated as follows: 1. 2. 3. Clamp 3 was closed and the clamp 1 on the inoculating side arm was opened. The inoculator (Fig. lb) was used to take up some Of the suspension of algae from one Of the subcultures. The tip Of the inoculator was pushed into the surgical rubber tubing of the inoculation side arm, and the squeeze bulb was used to force the solution well into the main air line. Clamp l was then replaced in a position immediately adjacent to the main air line and the inoculator was removed. Clamp 3 was Opened and the inoculation was swept into the Florence flask by the gas stream. Vigorous aeration was maintained until a dense culture was achieved. During this period the following points should be checked routinely: 1. 2. 3. h. 5. level of distilled water in the air line bubbler level of water in the constant temperature bath temperature of constant temperature bath drainage of exhaust line filters, which accumulate condensation rapidly proper speed of aeration When a culture was to be harvested, the following procedure was followed: h8 l. Clamp 5 was opened partially, clamp 3,was closed, and the connection at the nonsterile end Of the bacterial filter for the air inlet was broken. 2. The Florence flask, in the inverted position, was then raised out of the water bath with a simple, prearranged hoist and the drain clamp was Opened. Vigorous swirling at this time will clean Off any clinging algae. 3. About five and a half 1. of culture was drained out and the drain clamp was closed. The Florence flask was then replaced in operating position and the air line filter reconnected. h. The refilling side arm was connected to a 12 1. carboy of sterile media as follows: a. b. C. d. The surgical rubber tube and clamp (Fig. la) were slipped off. The refilling side arm was cut off just on the inside of clamp 2 and the end was immediately slipped over the glass tube on the media carboy. Medium was siphoned into the Florence flask until it was filled to within about one inch of the top, then clamp 2 was replaced on the refilling side arm and closed. Clamp 3 was Opened. The "starter" of culture left in the Florence flask from the previous culture rapidly multiplied and the whole process could be repeated many times without reinoculating the culture 0 By following the procedures above it has been possible to Obtain 50-100 ml. of packed cells per week. The apparatus has been maintained in continuous Operation for as long as six months at a time without loss of culture. When a culture was harvested, the volume of medium removed was recorded and the algae were centrifuged in an 3‘ Vie . ”w an. Al: tad In t. a. I. a» ..../ .1 .f .1 l. l . 'v'. ad? F _ 149 International refrigerated centrifuge, model PR-2, using 250 ml. buckets in the number 259 head. The cells were then resuspended in distilled water to wash Off the residual medium, and centrifuged in graduated glass tubes for six minutes at 3,000 r.p.m. in the number 82h head (1,770 g). The packed cell volume (p.c.v.) was then recorded and the cells were either utilized immediately or frozen until needed. The Mass, Synchronized Culture g£_Chlorella An agar slant culture of Chlorella pyrenoidosa van Niel 211 was obtained from Dr. R. R. Schmidt, Department of Biochemistry and Nutrition, Virginia Polytechnic Institute. The liquid medium formula, given below, was also obtained in a personal communication from Dr. Schmidt. The medium was made up and sterilized in 12 l. pyrex carboys (Figure la). The medium.was made up by dissolving 30 gm. KN03, 30 gm. KHZPOH, and three gm. Mg30u°7 H20 in eight 1. of dis- tilled water. Then 120 ml. of each of the four stock solu- tions listed below was added, the solution was diluted to 12 1. with distilled water and the pH was adjusted to 6.75 with two normal KOH solution. 1. micronutrient stock solution ll.u gm. H3B03 0.89 gm. (NHH)6M°7OZM' h H20 1.58 gm. CuSOu . 5 H20 8.82 gm. ZnSOh . 7 H20 50 0.h8 gm. COSOh . 7 H20 1.1m gm. MnC12 . 14 H20 dissolve in one 1. distilled water 2. acidified ferrous sulfate stock solution Dissolve 5.0 gm. FeSOh . 7 H20 in one 1. distilled zgigr and add 20 drOps of concentrated sulfuric 3. calcium chloride stock solution Dissolve 11.1 gm. Ca012 - 2 H20 in one 1. of distilled water. h. chelate stock solution 50 gm. EDTA (free acid) and 31 gm. KOH are brought to one 1. with distilled water. During subculturing it was found to be necessary for rapid growth to add one percent glucose to the usual liquid medium. This addition was suggested by the studies of Reisner (110). 50 ml. of medium.was added to each 125 ml. Erlenmeyer flask and an aerator containing a bacterial filter was inserted in a cotton plug. Then the subculture flasks were sterilized and inoculated with algae using a flamed wire. The subcultures were immersed in a water bath at 20-25° C. and aerated with compressed air. A 30 watt fluorescent light was used for illumination. Subcultures were used to inoculate a 650 ml. semi-mass culture apparatus which was illuminated by an immersed neon tube. This second-stage apparatus was aerated with one to five percent carbon dioxide in compressed air. It was maintained at 20-250 C. and was connected to a carboy of 51 sterilized medium. Before inoculation the apparatus was flushed with 70 percent alcohol and then several times with sterile medium. The apparatus used for mass synchronization was special- ly designed and constructed from three-eighths inch plexiglass in such a way that saturation light intensities could be maintained with dense cultures. The apparatus is diagrammed in Figure h. It consisted of three parallel compartments four cm. thick by 76 cm. square. The outer two contained baffles and were used as constant temperature baths. An American Instrument Company constant temperature circulator, number 701-h8l, was used to circulate distilled water at 25° 0. through these two compartments. The middle compart- ment was the actual algae chamber. It had an operating capacity of 20 l. Circulation was maintained by a flow of compressed air and five percent carbon dioxide from a series of small holes in a polyethylene tube, which was held across the bottom of the algae chamber. Two banks of AB inch, ho watt, daylight-type fluorescent lights provided the lighting. On one side a bank of seven tubes was adjusted so that the average light intensity on the algae chamber surface was about 800 foot-candles. 0n the other side a bank of six tubes was adjusted so that the average light intensity on the algae chamber surface was about 700 foot-candles. Thus by turning both banks on at the same time, an intensity of 1500 foot-candles could be 52 top view .3 l““*l 76 n- 33. 63. 631411913541 ”- _______g _._.._....._...._ bottom Figure 14., diagram of the plexiglass chamber of the mass, synchronized culture apparatus - _.. -4. ('9 .5. III I . .y‘ «by .h V .-d Ht .e‘.‘ .u. ‘ find 53 obtained. Light intensity was measured with a Weston illuminator meter, model 756. A siphon was used to remove the algae and, simultaneously, while this was going on, the algae were run through a Sharples continuous centrifuge, model M-hl-2h-8CYA3b, using a large Jet. Only twenty minutes were required to process 18 l. of algae suspension. The algae were then transferred to 100 ml. tubes and centrifuged in an International refrigerated centrifuge, model PR-Z, washed once with distilled water, and finally centrifuged again in the high-speed head, number 82h, at 3,600 r.p.m. for 10 minutes (2,120 g). The algae were then frozen until needed. Vblume of suspension harvested and packed cell volume (p.c.v.) of Chlorella were recorded for each harvest. The basic method in initiating synchrony in Chlorella has been reported by Schmidt (121) and further details have been received in a personal communication. In his work Schmidt reports a degree of synchrony of approximately 90-95 percent. No attempt was made in the present study to deter- mine percent synchrony, but visual microscopic checks were made at key times to determine whether the cultures were growing normally and whether daughter cell release occurred at the right time. In order to initiate synchrony, a culture would be grown in the second-stage apparatus described above and, after a rapidly dividing culture was achieved, it was allowed to grow until a cell density of about one ml. p.c.v. per 514 100 ml. of medium was reached. By frequent microscOpic checks a period could be found, during which about 80 percent of the culture was centrifuged at 10°C. and resuspended in fresh media. This cell suspension was then used to inoculate the algae chamber of the synchronization apparatus. This chamber had been previously flushed out with 70 percent alcohol followed by sterile medium. The dilute cell suspension in the synchronization chamber was then subjected to 800 foot-candles of light for 18 hours. The Chlorella were then darkened for 12 hours by turning the light banks off and covering the apparatus with several layers of black cloth. During this dark period the algae tend to accumulate at the young, daughter cell stage. Then the Chlorella were subjected to periods of 1500 foot- candles of light for 18 hours and 12 hours of darkness alternately as long as necessary to complete the desired experiment. After four light cycles, the algae were con- sidered adequately synchronized and harvests were begun. Usually only 15 or 16 l. were harvested so that only one cycle would be required before another harvest could be made. In his studies Schmidt (121) found most of the phos- phate uptake from the medium to occur between six and nine hours of light, and nuclear division to begin at about 1h hours. The synchronized Chlorella life cycle is shown dia- grammatically in Figure 5. Under the conditions described most mother cells were observed to release four daughter cells. In a- .- . 3T 4‘ A 914. ~ wwuw N;- Inhibition 2f Metabolism with Chloramphenicol In one case no parts per million (p.p.m.) of chloram- phenicol was added to the synchronization chamber after nine hours of light and the Chlorella were harvested after three additional hours of continued light period. The chloram- phenicol for this eXperiment was obtained through the courtesy of Parke, Davis and Company, Ann Arbor, Michigan. It was added as a solution of 100 mgm. of chloramphenicol in one 1. of fresh nutrient over a period of 15 minutes. This experi- ment and the concentrations used were suggested by the report of Takeda (132) that hO p.p.m. of chloramphenicol completely stepped net protein synthesis in Pseudomonas fluorescens, and the report of Brock (15) that 50 p.p.m. caused an immediate halt in protein synthesis and growth in Escherischia coli. Isolation and Characterization gf‘g Bacterium.From The Anabaena Culture Dense liquid cultures of the bacteria were obtained from liquid cultures of Anabaena by inoculating into flasks of the regular Anabaena medium which had been enriched with four gm. glucose, one gm. peptone, and one gm. of yeast ex- tract per liter. These flasks were then incubated at 30° C. in the dark with occasional swirling for 12 hours. Some of the liquid from these flasks was used to streak the surface of agar plates made by adding 15 gm. agar, eight gm. glucose, four gm. peptone, and four gm. yeast extract per 56 Ar“"——-1::"‘~“‘-. 6hr. fishr. total darkness —_— -—__d_—— ——— 1500 foot-candles 0hr.I(-—3-i-%§§§d fie—e 18 hr. 9hr.:‘ nuclear division 61119.. Oizhr. >\o/ ohloramphenicol 9th. (no p.p.m.) G 12 hr. Figure 5, synchronization cycle 8f_ghlorella ‘pyrenoidosa (Van Niel 211) at 25 C. .1 i w... .E .3. .3 t. .4 Mm .4 a .fi. s. .. . o G I y I.\ u h." h I! as. 9. v Inn-u ..\ t. . .0 n. a. uh Av. 57 liter to regular Anabaena medium. One type of colony dev- eloped. A Bacto Unidisk for antibiotics, high concentration, was then placed on the plate to test for sensitivity to various antibiotics. Isolations Fractionation of the Phosphorus Components of Anabaena Figure 6 outlines the fractionation procedure. The procedure, up to the crude preparation, was a slight modi- fication of the procedures reported by Kulaev (80) and Juni (63). The phenol extraction has been described by Gierer (h3). Standard Isolation g: the RNA-Polyphosphate g: Chlorella A frozen sample was thawed in 300 ml. of distilled water which was adjusted at intervals to pH 11.5 with KOH. After one hour at room temperature the pH was adjusted to 7.u with HCl and 300 m1. of cold, water-saturated phenol was added. The mixture was shaken for five minutes and then centrifuged for eight minutes at five degrees. The upper (aqueous) phase was saved, 300 ml. of water saturated phenol was added and the process repeated. The upper phase was then extracted four times with ether and evaporated under reduced pressure for a short time to remove the residual ether. One percent sodium acetate was dissolved in the solution, and one drop of 2 N HCl and four volumes of alcohol were 58 _.n_n___._a ested “W— extract h times with EtOH:ether (3:1) at room.temperature F P-lipids A‘ aciL-soluble ll“ extract 2 times, 30' at 2° with 5% TCA fraction flDD 5‘00 Ml. WAT filter, add 0.6 volume Kc.” To Ply/[6 conc. 0H and 0.1 ALLow To ST‘flA/D volume 0 15% MgCl , [HA9 AT 1004 stir and refrigeraée TEA/H? 4507mm! 2 hrs. esrén‘r‘ I X 1 acid-soluble l wash.with alkali-soluble residue organic-P 10% NHAOH, fraction add 30 ml. 1 N H01, ADD my! flCETIc ACID stir, can- T0 Pl, “geEm/éEKflTE +trifuge, [If/9. add 280 ml. "33h "1th add dil. R EtOH, re- 1 N H01 HCl to pH nucleo- frigerate discard 3.0, then protein overnight add 2 vols. EtOH, cool 7‘ ~ 11' ortho-P oligopoly-P discard Dissolve in wat aiding KOH to p 11, add dil. HC1 to pH . Carry t a t ith out 3 extractio with #2 ex r 0 W 1 vol. water-sa at d phenol L_ether, flash h, 1 t so 6 preparation p eno a ' evaporate the ‘water phase phenol phase (discard) Figure 6, fractionation of phosphorus couponents of Anabaena I"! ,2 H”: F. 59 added. The solution was stirred and more sodium acetate was added slowly until a flocculent precipitate formed. The solution was then put in a freezer for one hour with occas- ional stirring. The precipitate was centrifuged at 0°, washed with alcohol, and then dried at reduced pressure. The material thus obtained was dissolved in 30 ml. of 0.01 M tris buffer, pH 7.6 and was ready to be applied to a column of DEAE-cellulose. Preparation and Elution g£_Diethylaminoethyl Cellulose Columns The resin was placed in a large column and was washed with saturated NaCl solution, then with 0.1 N NaOH, and finally with a large volume of 0.01 M tris buffer, pH 7.6 until the pH of the eluate is 7.6. The columns used for the fractionation of RNA-polyphosphate were packed with only very gentle pressure. After the resin was packed it was washed with a small volume of buffer and the sample was applied. The column was then washed again with 20 ml. of buffer. In the case of the material from Anabaena, a column with a resin bed about 20 cm. tall and two cm. in diameter was used. In the case of the material from Chlorella, a resin bed about 20 cm. tall and one cm. in diameter was used. The columns were connected to a 500 ml. fixing flask and this was filled with 0.01 M tris, pH 7.6. The mixing flask was then connected to a reservoir to which a salt 60 solution was added. In the case of the work with Anabaena, 2 M.NaCl was added to the reservoir, and 10 ml. fractions were collected from the columns with a Rinco fraction col- lector. In the case of the work with Chlorella, a series of NaCl solutions were added to the reservoir: first 200 ml. of l M NaCl, then 200 m1. of 2 M‘NaCl, 200 m1. of 3 M NaCl, and finally 200 ml. of M NaCl. Five ml. fractions were collected from the columns with a Rinco fraction collector. Miscellaneous Chemical Determinations gytgchemical Location gf'Polyphosphate The location of polyphosphate in the Anabaena cells was carried out with toluidine blue by the technique re- ported by Keck (66). P32-Phosphate Incorporation In order to determine if the polyphosphate pool had a rapid turnover, 0.9 ml. of packed Anabaena cells were sus- pended in the usual liquid medium which had been diluted ten fold. The suspension was equilibrated at 30-35° C. with a 1000 watt flood-lamp. Occasional aeration was accomplished by bubbling a stream of air through the suspension. About 25 microcuries of P32-phosphate were added and after three hours the suspension was centrifuged. The supernatant and precipitate were boiled separately for 10 minutes. An aliquot of each was then chromatographed in both the two- .x V. I. 61 dimensional system of water-saturated phenol and butanol- propionic acid- water described by Benson (8) and the one- dimensional system for inorganic phosphates, described later. In both cases radioactive spots were located by authoradio- graphy using no-screen X-ray film. Phosphorus Total phosphorus was determined by the technique of King (68). Acid-labile phosphate-phosphorus was determined by hydrolyzing samples for seven minutes with one normal HCl in a boiling water bath and then determining orthOphosphate by the method of.Fiske and Subbarow (38). In all cases results are reported in terms of weights of phosphorus. Ribose and Deongibose Ribose was determdned by the orcinol reaction (25) using a no minute hydrolysis with 6 N HCl in a boiling water bath. A solution of adenylic acid was used as a standard. Deoxy- ribose was tested for by the diphenylamine reaction (21). Total Nucleic Acids Total nucleic acid was determined by the method of Webb (lhl). Protein gggm M Protein was determined by both the Lowry method (93), using bovine serum albumin as a standard, and by the ninhydrin reaction. The ninhydrin reaction was carried out by heating one ml. of sample with 0.2 ml. of a 0.1 percent solution of triketohydrindene hydrate in a boiling water bath. 62 Total Carbohydrates Total carbohydrates were determined by the indole reaction for total carbohydrates (25) using adenylic acid as a standard. Metachromasy Metachromasy was determined by a modification of the method of Damle (28). Two m1. of a solution of 30 mgm. toluidine blue in one 1. of distilled water was added to the sample and the volume was diluted to 10 ml. with distilled water. The maximum decrease in Optical density at 630 mu was measured with a Beckman, model B, spectrOphotometer and compared with a standard curve. This curve was constructed by carrying out the reaction with a series of concentrations of a synthetic polyphosphate obtained from.Mensanto Chemical Company, St. Louis, Missouri (marketed as sodium hexameta- phosphate). This polymer has been determined to have an average chain length of 16 phosphates. Care was always taken to make sure that the sample concentration was low enough to be on the ascending side of the standard curve. Ultraviolet Spectra Complete ultraviolet spectra between 220 mu and 3&0 mu were recorded using a Beckman, model DK 2, recording spectro- photometer and one cm. quartz cells. In cases where Optical densities at individual wave lengths were determined, a 63 Beckman, model DU, spectrophotometer and one cm. quartz cells were used. Infrared Spectra Samples of RN -polyphosphate complex were prepared in two ways. In the first an effort was made to avoid denatura- tion of the complex. Ten ml. of complex was dialyzed against 0.5 M KBr and the KBr solution was changed several times. In the second, a ten ml. sample was dialyzed exhaustively against distilled water to denature it and then it was dialyzed against 0.5 M KBr. Both samples were then lye- philyzed and stored in a desiccator., Aliquots of h00 mgm. of the powder were weighed out and used for pressing pellets in a hydrolic press. Infrared spectra of the pellets were obtained with a Beckman, model IR 5, recording spectrOphoto- meter. _§ualitative and Quantitative Nitrogen Base Determinations The first method involved the alkaline hydrolysis of RNA by incubation for 18 hours with 0.5 N KOH at too 0. This resulted in the free nucleotides. Subsequent neutral- ization with cold 36 percent HClOu resulted in the precipita- tion of KClOu which was filtered off. The filtrate was subjected to column chromatOgraphy by the method of Hurlbert (57). The separated nucleotide solutions were then lyo- philized to remove the formic acid. Ultraviolet spectra were determined at both acid and basic pH to verify their identities e 6h In a second method samples were hydrolyzed in sealed tubes with 1 N HCl for one hour in a boiling water bath as described by Smith (12h). This results in the free purine bases and the pyrimidine nucleotides. The hydrolyzed material was then chromatographed on Whatman number one paper by the descending technique using the isobutyric acid-NHuOH-water (66/1/33) solvent described in circular 0R-10, Pabst Laboratories, Milwaukee, Wisconsin. Known standards were also chromatOgraphed to determine Rf values. In the column chromatOgraphy when base ratios were calculated, the areas under the 260 mu optical density (0. D.) elution curves and the extinction coefficients published by Pabst Laboratories in circular 0R-10 were used. In the case of paper chromatography, the spots were eluted by immersing the cut out spots in five ml. of 0.1 N HCl for one hour and measuring the 0. D. at 257 mu for adenine, 256 mu for guanine, 280 mu for cytidylic acid, and 262 mu for uridylic acid. Base ratios were calculated using the extinction coefficients in Pabst Laboratories circular OR-lO. Esp E’ChromatOgraggy g: Pglyghosphates A modification of the system reported by Thilo (137) was used. The solvent was prepared by adding 20 ml. of 20 percent trichloroacetic acid and 10 ml. of water to 70 ml. of isopropyl alcohol, and then titrating to pH h.0 with concentrated NHhOH. Whatman No. 1 paper was used and the “hi. ‘I”. I!“ led .u . n to“ c~ P: H. Was ‘0 y‘C 65 chromatograms were developed by the descending technique for 18 hours at room temperature. Spots were located by spraying the paper with Hanes- Isherwood reagent which was composed of five ml. of 60 percent H010“, 10 ml. 1 N HCl, 25 ml. four percent ammonium molybdate, and 60 ml. of water. The sprayed paper was placed in an air oven at about 70° C. for several minutes and was then exposed to ultraviolet light for several minutes to develop the spots. Preparation and Use of Charcoal Norite was treated by exhaustive washing with 0.1 N HCl and then, as suggested by Lisa (89), with a large volume of four percent potassium-EDTA at pH 7.0. Then the charcoal was washed with 0.01 M tris, pH 7.6 to which one percent NaCl had been added. In each adsorption study about three gm. of charcoal was added to between 5 and 10 ml. of sample and the mixture was stirred occasionally for 20 minutes. Then the mixture was filtered with a Bachner funnel and a vacuum flask, recycling the filtrate several times. Enzymes Crystalline ribonuclease was obtained from.Worthington Biochemical Corporation, Freehold, New Jersey. Snake venom phosphodiesterase was obtained through the r —_ . I ‘ .- . v‘y'd v- £1: M“ .u‘ dvl 66 courtesy of Dr. Charles Mead, who isolated the enzyme free of 5'-nucleotidase activity from Crotalus terrificus venom. LyOphilyzed Crotalus terrificus venom was obtained from Ross Allen's Reptile Institute, Silver Springs, Florida. A polyphosphatase complex was isolated from dried brewer's yeast, obtained from Budweiser Breweries, Inc., St. Louis, Missouri. The isolation used was reported by Mattenheimer (99) and the 20-70 percent ammonium sulfate fraction was used for the studies to be reported. Specific reaction conditions for each enzyme are reported with the individual sets of experimental results. RESULTS AND DISCUSSION Anabaena Distribution 2: Phosphorus When cultures of various ages were extracted first with alcohol:ether (3:1) and then with KOH at a pH of 10, the data in Table I was obtained. It can be seen that although the total phosphorus remained about the same, the percent which was in the form of orthophosphate declined sharply with older cultures. The results of two fractionations of the phosphorus compounds of Anabaena are shown in Table II. In both cases polyphosphates constitute about hO-SO percent of the total phosphorus. The bulk of the remaining phosphorus was ortho- phosphate. Incquoration g: P32-Phosphate P32-1abeled phosphate was utilized to determine whether the polyphosphate had a rapid turnover. After three hours incubation no labeled phosphorus compounds other than ortho- phosphate could be found in the medium. Upon paper chromato- raphy of the hot-water extract of the cells, a small amount of radioactivity was found in sugar-phosphates, AMP, ADP, and orthophosphate. The origin, which is occupied by polyphosphate in both systems, was very radioactive. 67 68 Table I. Changes in the orthophosphate content of the alkali-soluble fraction of Anabaena w total-P ortho-P % (gifugfcégg7lf) (ug.P/ml. p.c.v.) (ug.P/ml. p.c.v.) ortho-P 0.5 598 598 100 1.8 565 14,85 86 7.8 Sho 2&0 uh * p.c.v. = packed cell volume Table II. harvested at two culture densities 69 Distribution of phosphorus in Anabaena culture # 1 culture # 2 (density =/5.6 1.) (density = 12 ml. fraction m1. p.c.v. p.c.v. 1.) ug.P/ml. p.c.v, % ug.P/ml. p.c.v. % P-lipids 2 0.3 8 1 .1; acid- soluble 68 10.2 5 0.9 organic-P residue 29 h.3 77 13.1 ortho-P 22h 33.5 175 29.9 oligo- poly-P 130 19.5 153 26.1 RNA-poly-P (crude 21E 32.5 168 28.7 prep) (13 )* (20.2)* total 668 586 a- figures in parenthesis are A7-P values 70 These data, although not conclusive, indicated that Anabaena exhibits the same type phosphate metabolism as Aspergillus (80) and brewer's yeast (8h). Since no radio- active ATP was detected it seems plausible to assume that newly synthesized ATP was being utilized rapidly in poly- phosphate synthesis. Location g£_Polyph03phate ig_thg Cell The metachromasy of Anabaena cells was found to be located completely within the pseudovacuoles, whether the cells were fixed before staining or not, This ability to give the metachromatic reaction could be destroyed by exhaustive extraction with ice-cold 10 percent trichloro- acetic acid. The reactivity was not removed by short treat- ment in 0.1 N H01, indicating that the reaction was not due to nucleic acids or to sulfonated polysaccharides. However, as a result of further studies reported below it is likely that this technique locates only free polyphosphates or denatured RNA-polyphosphate since native complexes give no metachromatic reaction. Thus their location in the cell is still unknown. Characteristics gf the Bacterium Isolated from the Culture This bacterium was found to be nonmotile, rod-shaped, and about one micron long. It was gram-negative and was strongly inhibited by such tetracycline antibiotics as aureomycin, terramycin, and tetramycin. It was not inhibited “T“— .Ac 5: is ‘5 V‘ 71 noticeably by penicillin, chloromycetin, polymyxin, erythro- mycin, or distreptomycin. Characteristics 2f RNA-Polyphosphate Phenol Preparations Data obtained from a series of phenol preparations is recorded in Table III. In addition, protein was determined on some phenol preparations. Unless great care was taken to remove every trace of phenol the protein values were erratic, since phenol itself gives a positive reaction with the Lowry reagent. In a few cases protein was determined to be either absent or only present in such small amounts as to be insig- nificant. A rough correspondence can be seen within experi- ments between difference phosphorus and nucleic acid phosphorus, as determined by multiplying total nucleic acid values by a gravimetric factor. A similar correspondence occurs between ribose as determined with the orcinol reaction and ribose as calculated by multiplying difference phosphorus by the appropriate gravimetric factor. However, a considerable variability between experiments was obtained in all of the parameters. This fact indicated that a variation in the composition of the harvested Anabaena existed. In two cases crude preparations of RNA-polyphosphate were tested for deoxyribose and none was found. One residue fraction was tested and found to contain 17 ug. deoxyribose per ml. p.c.v. It should be remembered that this test is only given by the purine deoxynucleotides. When acid hydrolysates of crude preparations of RNA- Table III e 72 of RNA-polyphosphate from.Anabaena Parameters of typical phenol preparations experiment parameter 1 2 3 L. 5 6 7 8 total-P 95 6h 83 kl 216 116 h51 77 (ug.P/ml. p.c.v.) A -P 81 ho 71 33 131+ 61 366 52 (ug.P; . p.c.v.) difference-P 1h 2h 12 8 82 55 85 25 (IlgeP/mle p.c.V.) (A7-P)/(diff.-P) 5.8 1.6 6.0 3.9 1.7 1.1 u.3 2.1 (diff.-P)(150/31) 67 - 60 no - - - - _ ribose 105 - 69 ha - - - - (Ilse/ml. proVO) tota nuc. acid - - 210 56 1060 272 1,380 228 (ug. ml. p.c.v.) (nuc. 801d)(:%;3) - - 20 8 103 26 13h 22 0.D @ 260 mu 6.5. a 286 mu specific metachromasy 1.h3 1.2M 1.92 1.37 1251 1.8 2.7 1.5 1.1 1.1.68 1.h1 l.h 73 polyphosphate were separated by paper chromatography, adenine, guanine, cytidylic acid, and uridylic acid were identified. No thymidylic acid was detected. When an aliquot of the phenol preparation used in experi- ment 3. Table III, was subjected to alkaline hydrolysis and the nucleotides were resolved by ion-exchange chromatography, the data in Tables IV’and V were obtained. When consider- ing these results it is important to remember that under the hydrolysis conditions used, RNA yields 2' and 3' nucleotides and very little hydrolysis of polyphosphate occurs. The latter is illustrated by the synthetic polyphosphate control, which still had a high Specific metachromasy after hydrolysis. No nucleoside di- or triphosphates were detected but any nucleoside phosphates with higher degrees of phosphorylation would probably not have been eluted from the resin. Refer- ence to the significance of these results will be made in later sections. Changes Occurring 33 Cultures Reagh Higher Densities The variation in the preperties and amounts of RNA- polyphosphate in phenol preparations could be caused by a number of factors. Of these the one that seemed the most likely was the change in light intensity due to shading in older cultures. This effect could have been caused either by influencing the types and speed of metabolism in the cells, or indirectly by causing an accumulation of cells at some critical energy-requiring stage of the life cycle. The first of these possibilities is suggested by 7h Table IV. Nucleotide composition of the alkaline hydrolysate of an Anabaena RNA-polyphosphate phenol preparation nucleotide mole percent CMP 22.8 AMP 20.9 GMP 31.6 UMP 2A.? Table V. Effects of mild alkaline hydrolysis on an RNA-polyphosphate phenol preparation and on synthetic polyphosphate specific % r metachromasy Ay-P) - (ortho-P) RNAepolyphosphate ' before hydrolysis 1.5 85.5 RNA-polyphosphate after hydrolysis 0.1 8h.5 synthetic poly-P before hydrolysis 1.2 - synthetic poly-P after hydrolysis 0.9 - .| r: .V‘. n!» .. 75 changes in the reapiration rate. Brown (16) found the respiration rate to change abruptly in Anabaena.upon expo- sure to light after a dark period and Sorokin (126) showed a light effect on the rate of respiration in Chlorella, independent of the changes induced by the life cycle. The second possibility is the basis of present methods for obtaining synchronized algae cultures. One of the early reports was that of Tamiya (133). He induced synchrony in Chlorella by allowing a culture to become very dense. About 80 percent of these cells accumulated at the "nascent" young daughter-cell stage. Figure 7 illustrates the increase in packed cell volume and percent of cells in a visible state of cell division with time in a typical culture. The increase in packed cell volume after nine or ten days is largely a reflection of the production of an excessive amount of sheath material encas- ing the filaments. The accumulation of up to 80 percent of the cells in a state of cell division indicates that this could be a factor in the observed variation of isolated RNA-polyphosphate. It could also happen that such a dense culture, when diluted after a harvest, might retain some degree of synchrony for several days and thus give erratic results in the case of algae harvested within that period. Changes i2 RNA-Polyphosphate with Increasing Culture Density Phenol preparations were isolated from a series of cul- tures of different densities. These phenol preparations 76 Figure 7. Changes occurring in a typical Anabaena population during growth 77 “ITIOIIOIV assessed .H\.>.o.n .du O 7 8 2 3 2 .m. .12.. l H 1 9 6 3 0 q - q u - u) 9 q . A . . Ln L 19“” a L d n 7 .1.— .7 e e . We m. .1 .5e F I w / t I l u c 3 l n . p b b - u r L 5 JV 0 0 0 0 0 0 0 0 m 9 8 7 6 5 .4 3 m m 0 “Illelv genesis fine we owe». a??? 3 afloe m 78 were then fractionated on DEAE-cellulose columns. A number of eluate areas containing RNA and polyphosphate were separated by this technique. The elution pattern as measured by the optical densities at 260 mu. and by total phosphorus are shown in Figures 8-lh. The amount of polyphosphate in Figure 8 is much greater than in Figure 1b. In Figure 1h there is also a large RNA peak, which apparently contains very little polyphosphate. One very interesting result of calculations made from these curves is illustrated in Figure 15. The culture density series could be broken up into three parts. In the first part there was an excessive amount of polyphosphate, and even though there was also a relatively large amount of RNA, the ratio of O. D. units of RNA to mgm. of total phos- phate was very small. This low ratio was characteristic of young cultures. In the second part this ratio was fairly stabile between 16 and 21. Finally in the third part, composed only of the oldest of cultures, the ratio increases rapidly due to an increase in the amount of RNA. An interpretation of these results will be discussed in a later section with similar data from Chlorella. The specific metachromasies and ultraviolet Spectra of a series of tubes from each elution were determined before and after exhaustive dialysis (2h hours) against distilled water. In this case specific metachromasy was determined by dividing the metachromasy by total phosphorus. The 79 Figures 8 - 1h. Elution patterns of acid- insoluble RNA and phosphorus from Anabaena) cultures of various densities (solid line: ug. total-P/ml./10 ml. p.c.v.; dotted lines optical density at 260 mu/lO ml. p.c.v.) 80 'Im/d-Imoq °Sn 85.32: .8 no»: .89 since woe—odes .HE 01m n Random 0.39.3.0 em shaman hangs one» em 2. 8 cm as on on S . o . fl 4 J 1 4 . q 4 d 00 o \‘8‘. .\ H II ’ ‘\ J H000 .” \O N If \ l e I/ \ m r 2., u 8.0 It. \ I .\ ’0 e\.\\ 3 a. \ I +3.0 \\ I mr 2). ~\ .. more 6 .. .. A5.6 N. _l .1 FOOD Q I. J @000 ms 092 43 'CI '0 finance 90 nopHH Rom mHHoo coxosm .HE N.> n haemaop oASPHSO .o shamam Menard can» (fi 81 'Iw/d-Ivzoq °3n .d' In me me mm m: mm mm ma m 00.0 a . . _ 1 No.0 - No.0 4 no.0 nm 092 43 '0 '0 82 'Iw/d-quoq °3n 05.320 no he»: hog 0.300 03309 .d: 06 n meanest 0.33.90 .OH 0.33% .3355 on?» 00.0 a0.0 «0.0 no.0 30.0 m0.0 00.0 50.0 00.0 nu: 092 4'9 '0: '0 83 “rm/d-Imoa 'Sn _Esaoos ho nopfia aoa mHHoo memosm .HE N.HH u huamdop canvass .aa cadwam homes: can» 00 00 em 0: 0m 0m 0H m u . .1 q “M‘Lqull 00.0 0“ \|!\1\\\ n H000 4 No.0 1 M000 .. 00.0 1 m0.o n 00.0 n 3.0 1 00.0 “m 092 43 “G ’0 Spence mo panda hem uHHoo deacon .da ma." huannoc casuaso .NH shaman aonasa can» om 0: on om 00.0 q - 8h ‘Im/d~I34°4 'Sn Hooo No.0 no.0 00.0 mo.o 00.0 h0g0 1 00.0 nm 098 43 ’G '0 ug.—60E no 90»: 90A 0.300 000:0an .HS mm H huamnou 0.39.30 .9. 0.3m?“ hon—gauche.» 85 'Im/d-I3404 °3n on 00 om 0: on om 0H H . . _ T . d ii1)\4000 ‘0“ I. '0’... 08"“:8 I... '. t 1 No.0 nm 092 4'9 'CI '0 86 'Im/a-Ivaon 'Sn 0“ E9358 .Ho 909.3“ you 9300 @0339 .HS 0.9.“. u huamaov 0.33.50 2.: mgm.—rm ow pagans and.» 0H oo.o Ho.o mo.o mo.o 10.0 mo.o 8.0 No.0 mooo nm 092 43 '0 '0 87 Figure 15. Total acid-insoluble RNA and phosphorus in Anabaena cultures of different densities (one 0. D. unit of RNA is equivalent to one m1. of solution with an absorbence of 1.0 at 260 mu in a one cm. cell) 88 TTarllarv .>.o.a .2 SEASS .swa no 7. lo a) L4 «J as 1* n. o, no 7. ,o a) L4 2; 9“ 1. Au 0 O O O C O O O O O O O O O O C O O O .l 1. .1 1. 1. 1i 1. 1. 1* no no no .0 n. no no no no nu . . q . . ‘1. . u . . . a q dig ... Mu; [I a) lad lo 9. :2 1.; nnu 1 \ 15 m . P n . p . . kw - b m“ p xh . . . .. .J ,b ?_ no no ,0 a. no no Au 9. Au no In .4 2; a» 2, a; 9. ac mm 9. 9. 1. 1. um 1. 1i h» .u 317$ .Es .2 23a .3 Es a .0 ty 1. nutrient) culture densi Figure 15 (m1. p.c.v./ Table VI 0 disstilled water in the metachromasy and ultraviolet absorption of’ separated areas from the elution patterns in Figures 8-11 89 Changes induced by exhaustive dialysis against specific metachromasy 0.D.O 260 mu 0.D.@ 260 mu Fig. tfibe . . mu before after before after before after dial. dial. dial. dial. dial. dial. 8 25 0.65 1.2 0.032 0.119 1.8 1.1 " 32 0.95 1.5 0.225 0.310 2.0 1.3 " 36 0.8 1.7 0.201 0.220 2.1 1.6 " 12 0.6 1.5 0.271 0.307 2.1 1.6 " 18 0.15 1.3 0.501 0.576 2.2 1.6 " 56 0.13 1. 0.160 0.211 2.1 1.5 9 26 0.38 1.3 0.026 0.068 1.2 1.1 " 2 0.33 1. 0.291 0.100 1.8 1.1 " 0 0.29 1.1 0.133 0.226 1.9 1.1 " 13 0.17 1.3 0.199 0.621 2.0 1. " 52 0.15 1.1 0.121 0.192 1.6 1.1 10 17 0.11 0.1 0.080 0.101 0.9 1.2 " 25 0.31 1.1 0.079 0.095 1.2 1.3 " 28 0.50 1.1 0.278 0.296 1.6 1.5 " 32 0.11 1.2. 0.311 0.301 1.9 1.8 " 35 0.22 1.1 0.311 0.307 1.8 1.7 " 9 0.02 1.2 0.219 0.261 2.0 1.6 " E1 0.00 1.1 0.112 0.176 1.9 1.7 " 3 0.00 1.2 0.198 0.209 2.0 1.7 “ 71 0.83 1.3 0.083 0.096 2.0 1.7 11 29 0.11 1.0 0.122 0.217 1.5 1.1 " 6 1.00 1.0 0.090 0.193 1.1 1.3 " 1 1.20 1.8 0.096 0.183 1.7 1.3 " 11 0.10 1.1 0.067 0.166 1.7 1.3 12 15 0.05 0.5 0.193 0.118 2.1 1.2 " 20 0.18 1.1 0.227 0.321 1.8 1.3 13 1’4, - " 0.0 5 0.216 1. 1.1 " 27 - - 0.090 0.221 1.1 1.2 11 a: 1.90 - 0.078 - - - 1080 - 1.090 - C- - 90 results are given in Table VI. No consistent correlation could be seen between the increases in specific metachromasy, which were observed, and the increases in 260 mu optical densities. A few generalizations can be made about the ultraviolet spectra. The spectra varied all the way from those in which the maximum was at about 258 mu and a deep valley was pres- ent in the region of 230 mu to those in which no valley _, us. Iain-n1 ..— riy’.’ occurred at 230 mu and only a plateau could be observed in the region from 250 to 270 mu. In general dialysis brought about a decrease in the 230/260 and 260/280 ratios. The 3 increase in O. D. at 260 mu upon dialysis was usually much f-f greater in those samples whose spectra lack a 230 mu valley. In some cases this increase was at least four-fold. Another characteristic of these spectra was a very rapid increase in optical density in the low wave length end of the spectrum. Such ultraviolet spectra and changes in the extinction coefficents at 260 mu could be explained on the basis of the assumption that a complex between RNA and some other compon- ent was being broken by dialysis. This breakage would have to be irreversible since addition of NaCl to the dialyzed samples only caused minor changes. Blout (10) has reported this type and magnitude of spectral changes in calf thymus DNA when it is mixed with small amounts of plasma albumin. The changes observed could not be explained on the basis of the hyperchromic effects resulting from the denaturating of the hydrOgen-bond system of the RNA, since such effects are 91 not of this magnitude. Further Characterization of the Fractionated RNA-Polyphosphate In one case a series of seven tubes at intervals in the elution from a DEAE-cellulose column were tested for ninhydrin reactivity before and after hydrolysis for one hour in 2 N H01. No ninhydrin reaction could be detected in any of the tubes, although the same amount of 0.001 M alanine was easily detected. This indicated that the phenol extraction had completely removed all peptides and proteins. A series of ribose determinations on elution tubes from one column are shown in Table VII. It can be seen that the amount of orcinol-positive material was about the same before and after dialysis. In Table VIII a series of total carbohydrate and ribose determinations are recorded from the elution tubes of another column. These carbohydrate analyses may be too high, because small particles of the DEAE-cellulose sometimes elute from the column and cause an increase in the total carbohydrate values. Therefore it is not known whether polysaccharides in small amounts are involved in the RNA-polyphosphate, as it is isolated. In the case of one column, five groups of tubes were Pooled and used to determine base ratios. The values in Table IX are the averages of paired determinations. No consistent changes could be seen. 92 Table VII. Effect of dialysis upon the ribose content of the eluates from.a DEAE-cellulose column tube ug. ribose/ml. # before dial. after dial. 12 1.3 1.3 18 6.0 1.8 25 1.8 5.0 31 8.7 10.6 36 6.6 3.2 Table VIII. Concentration of ribose and total carbohydrates in a series of elution tubes from a DEAE-cellulose column tube ribose total carbohydrates 1 # (ug./ml.) (ug./ml. as ribose) 13 0.6 12.5 50 3.0 1.7 53 2.3 12.7 57 2.5 2.2 62 3.5 5.5 70 5.0 12.0 k! --.._-__ ”man-n . .-- l l 93 Table IX. Approximate base composition of a series of areas of RNA from a DEAE-cellulose column WI mole percent tube numbers adenine guanine 0MP UMP 10-12 35.8 22.5 12.7 28.9 18-20 29.5 17.9 21.2 31.1 21-26 27.6 25.5 18.1 28.6 29-31 31.? 31.0 16.0 21.2 35-37 28.8 29.8 13.6 27.8 9h Stability of RNA-Polyphosphates It has already been noted that most samples show greatly increased specific metachromasy after dialysis against distilled water. The release of this metachromasy was utiliz- ed in a series of studies on the stability of the RNA- polyphosphate complexes, after purification on a DEAR-cellulose column. The results are reported in Table X. Nuld acid or u -r-mn - ’3 5.1.22” mild alkaline hydrolysis resulted in decreased metachromasy. Incubation with ethylene-diamine-tetraacetic acid (EDTA) did i not significantly increase the metachromasy. A most inter- 1 esting fact was the failure of boiling temperatures to j 11'. release the metachromasy, since such treatment will cause the complete separation of the hydrogen-bond systems of native DNA and synthetic RNA double helices. Alkaline pH's such as used in the extraction procedure would also have completely denatured ordinary RNA (30). The action of crystalline RNase on synthetic poly- phOSphate, yeast RNA, and both "native" and dialyzed RNA- polyphosphate was measured by the effects on metachromasy as presented in Table XI. Aliquots of experiments 2, 3, 7, and 9 were chromatographed, but no release of any free orthOphosphate or olig0polyphosphates (n< 7) was detectable. Aliquots of experiments 1, S, 7, and 9 were chromato- graphed to see if any free nucleotides or oligonucleotides were released. In experiment 1 none were detected. In experiments 5, 7, and 9 ultraviolet-absorbing material was 95 released, which had a small Rf and was probably a di- or trinucleotide. In experiment 5 a faint spot was detected with an Rf of about 0.7, which would be characteristic of a nucleotide. In summary, RNase seemed to attack both yeast-RNA and RNA-polyphosphate complexes whether dialyzed or not, but the enzyme did not affect synthetic polyphosphate. In the case of both RNA—polyphosphate and dialyzed RNA-polyphosphate, free low-molecular-weight polyphosphates were not released but some low-molecular-weight oligonucleotides were released and the Specific metachromasy was changed significantly. i J 1:“-. More will be said later about the interpretation of these data. Attempts tg_Produce Synthetic 23 Artificial RNA-Polyphosphate Complexes An effort was made to determine whether RNA-polyphosphate complexes were artifacts. This possibility was not likely, since the changes produced by dialysis against distilled water were not reversible. No effect; could be observed on the optical density at 260 mu of solutions of yeast-RNA or salmon-sperm DNA, when synthetic polyphosphate was added. These experiments were done in distilled water and in 2 M NaCl. In another eXperiment RNA was isolated from tobacco leaves by the same method as used in isolating RNA-polyphosphate from Anabaena, but synthetic polyphosphate was added at the beginning of the 96 Table X. Effects of various treatments on the specific metachromasy of Anabaena RNA-polyphosphate treatment specific metachromasy none 0.20 dialysis against distilled watafi 1.13 10 min. boiling 0.18 30 min. incubation with Na.EDTA 0.23 12 hrs. in 0.5 N NaOH, room temp. 0.09 10 min. boiling in 005 N NaOH 0.03 2 min. boiling in 0.1 N H01 0.05 30 min. boiling in 0.1 N H01 0.15 5 min. in 1 N H01, room temp. 0.09 Table XI 0 yeast-RNA, assay: 97 Effects of RNase on free polyphosphate, and RNA-polyphosphate 5 ml. substrate and 1 ml. of enzyme or buffer; incubate 30 min. at 25°; boil 5 min. substrates: Enough yeast-RNA was dissolved in 0.5 M tris, pH 7.6,to have an 0. D. @ 260 mu of 1.0. For synthetic polyphosphate 33 mgm. sodium.hexametaphosphate per 100 ml. 0.5 M tris at pH 7.6 was used. enzyme solution: 1.3 mgm. crystalline RNase in 10 ml. 0.5 M tris, pH 7.6 experiment substrate enzyme specific metachromasy 1 buffer + 0.0 2 synthetic polyphosphate - 1.0 3 synthetic polyphosphate + 1.0 h yeast RNA - 0.0 5 yeast RNA + 0.0 6 RNA-polyphosphate - 0.1 7 RNA-polyphosphate + 0.2 8 dialyzed RNA-poly-P - 1.15 9 dialyzed RNA-poly-P + 0.6 98 alkaline extraction step (Figure 6). The resulting phenol preparation was then fractionated on a DEAE-cellulose column and no material could be found which exhibited the properties of the RNA-polyphosphate complexes isolated from Anabaena. Chlorella Changes 1g RNA-Polyphosphate During the Life Cycle Samples of synchronized Chlorella, taken at three—hour intervals in their life cycle, (Figure 5), were used to isolate and fractionate RNA-polyphosphate, (Figures 16-25). In addition the effects produced by 21 hours of light are HR} given in Figure 26. Figure 27 illustrates the result N obtained when chloramphenicol was added after nine hours of light and the Chlorella were harvested three hours later. —— "‘11-! ‘1‘ . a. ho ‘ 'i 9&1. Six elution areas of RNA could be distinguished, since Y" they eluted at about the same places from column to cdlumn and their magnitudes showed systematic rather than erratic changes. Boundaries for these six areas were arbitrarily established and the areas were labeled A through F as can be seen in Figures 16-27. Six areas of polyphosphate, which roughly correspond to the six RNA areas, were also delineated and labeled I through VI. Studies and calculations were then carried out of the properties, amounts, and changes in these areas during the course of the life cycle. Figure 28 shows the changes in amounts of total phos- phorus and total RNA. A striking correspondence between the changes in these two components can be seen. This could be interpretted as evidence that the synthesis and utiliza- tion of polyphosphate and RNA are somehow interrelated. These points at which this relationship was not very close 99 100 Figures 16-27. Elution patterns of total RNA and phosphorus from synchronized Chlorella cultures at various times in the life cycle (solid line: ug. total-P/ml./10 ml. p.c.v.; dotted line = optical density at 260 mu/lO ml. p.c.v.; A—F= RNA areas, I-VI = polyphosphate areas) 101 'Iw/a-Ivnou '9n Meagan 035p oma cad ooa oo om o» oo om c: on om oa H O a u . . a d - \‘3Lous‘ .t .Nl‘lsl j! \‘l‘li’eiill I L oa I s s I], a m o s .1 s » . ON .I )1 N f‘& l J» K on I a a I (x o: I I om I 06 I I oa I I cm I pamHH an meson 0 .6a oeswaa o.o H.o «.6 m.o :.o m.o w.o To w.o 0.0 o.H H.H run 093 4’3 'CI '0 102 °tmya-Iazos 'Bn 11.2.2111 Ii named an unsqa.m .NH oaswaa hogan 0&5 ooa cm 0% ow oo om c: on om OH H . - I does — cries“. loosely 00‘ I \O I C. 6 es. 0 1 H00 ooo '\ I ~.o I m.o I 3.0 ~ ‘ - ~h-‘~~ I moo I. 0.0 I 5.0 I m5 I. 0.0 TI. 57.1.11: an: 092 43 ’CI '0 103 'Im/a-Ivnoq 'Sn o o o o o o {a c: o «a F- \o In ;t F\ .4 o I I I I I I I I (u Fl ' o 1‘9 r1 I .:.r 0 nm 092 43 ‘G '0 1 0- 9- 0.8 - 7.. 0.6- 0.5- 30 10 SO 60 70 80 90 100 tube number 20 Figure 18, 6 hours in light 101 'Im/a-Ivsos '90 O OH ON on 3 a co 2. om ONH OHH OOH om. cm a u d d pawn 5 6.85: o .3 95mg .3955 0&5 2. 8 cm 0: - - o.o do «.0 mac :6 m6 06 5.0 m3 moo o; H...” run 092 4'3 '(I '0 105 'Im/a-Iaaoa °3n pnwfla ad mason NH .om onswdm hogan ooh» QNH OHH ooH om cm on co om o: om om oa H o {:81— Sooootllo . . . . . :33.‘ OH I. 0’00 ll 0 h\0000 L 0:... N I om r N I . .. on a . I 2 ‘ I C 0.: T I om u 00 I I 2. r P) _ .. —|(\IH>||IIII+IIIAP >H:\+»HHH HHIJ H1; . cm I. 1 TL}? 10 a: a.o m.o m.o m.o 0.0 ~.o w.o ¢.o o.H HoH run 092 1V3 '(I ’0 106 'tm/a-Ianoa 'Sn quN P‘ . :55! F— pnwfia ca mason ma .HN onzmfim 9355 09.3 oHH ooa om om 05 oo om o: om om oa a d W 0H r om u om_‘ o:.. om.r ON I omfi. W 44 I I I 1 d 0)” |>1wd¢Iluu| uh 111 11.... ‘0: 0.. .0000. u - C — L ‘ Q g - l M x .r .. 1 t o to o 000‘ l l J l J o.o H.o «.0 m.o :.o m.0 o.o 5.0 m.o m.o o.H H.a m 092 43 'G '0 107 °tm/&-Iaaoz °3n ca cm on 0: am co 2. cm unwaa a4 mason ma .mm onsmdm .3954“ ooh» own oHH cod oo om op om cm 0: an em oH H ithogionIQoQII-CCI. q 1 fl 4 d 0.00.00... QO‘QJJ ‘0. r m. c. .....J ~ n ~ — J I. — l _ u — h I r __ n. - r u _ I .. . _ .. u _ .“ r a. . r. _ k H.o ~.o m6 :.o m.o o.o To moo m6 o.a H...” mu 092 43 'CI '0 108 'Iu/a-quoz °Sn o oa om on o: om co ow om oma oHH ooa oo om op It f‘ . . . I a!“ ‘SZ‘PQ ' 5? .ummwo an anaqn;m .mm onswfim hogadn can» ooo Hgo No0 moo :5 ma 0.0 woo w.o 0.0 ood Hod am 093 43 'G '0 Wfih‘ ‘ 110 'tw/J-Ianoa 'Sn a O O O 0 <3 in .d' M 1 O (s C Q mama ma mnzon 0 .mm «Aswan ho 955 09.3 LL o.o H.o m.o n.o goo m.o c6 ~.o 0.0 0.0 o.a Hod um 09? 4'3 ’CI ’0 st. 110 ’Im/J'I9404 °8n gnaw aH mason 0 .mm onsmHm .3 955 0&5 omH oHH ooH om om o~,. oo om 0: cm om oH H . o i‘woOoflblri 4 q 1 i q .::s: 10.000 .00 \DOQ J OH I O... .— 59 Q: 0 A .I am I» l f . o a O L ’00 g ‘5 om: an . 1 ._ ._ 3 1 . ~ .. . _ 1 a - . u w H .. a .. ..... - o~_u n T>+\I>H +HHH+HH+IHI+ I om.. Thu/\ITmi‘ni‘i ij .. o.o H.o «.0 m.o :.o m.o coo ~.o m.o m.o o.H H...” nm 098 4'3 ’CI '0 111 .Tq/&.Iaqoq ‘3“ ONH ow hogan and». 00 pawHH nH mason Hm .om onsmHm OH! ON! on . om I 00! ow I on I oHH 00H om ow < - 1 A1 A. .4 q 0 out... ciclnll‘OOtoOoIOOcco 0". . .cO I. 00 T: ‘- om o.o H.o N.o m.o :.o m.o o.o 5.0 nm 092 a? 'G '0 woo 0.0 Din o ow ONH 0.: OOH I om om ON udoanpfin on» on HongofieanoHno €370 0: .8 83300 on» .898 33H .5 9300 32: m :23 05 panH 3 0.33 0 .~.« 93me .3253 cab» 00 om 0.0 H.0 «.0 m5 0.0 m.0 0.0 To o6 0.0 0.H an nm 093 4'9 'CI ’0 i 113 Figure 28. Total RNA and total phosphorus per 10 m1. p.c.v. of Chlorella during synchronous growth (4321 hours in light instead of dark- ening at 18 hours, = 140 p.p.En. chloramphenicol added at nine hours in light) 11h (OUTI pIIOS) s-Iaqoq 'mfim HO Fl F1 0‘ «D b— {J U\ ;1 ex on I U l I l I ' r 1 I I J ,r d’ 1’ ‘x:z’ \\ ‘\ ‘\ \ \ \II «I /f x0e 1’ 1’ ‘\ ‘\ ‘\ \tL\’ o \ ‘\~ ‘\ ‘x \ l l 1 I I 1 ., :1 l 29:: ssmssza rs 180 3. -0‘ 1 hours in dark 4: 1 9 hours in light eurt uefloaq) VNH JO Satan Figure 28 115 are also interesting. At six hours of light the synthesis of RNA was somewhat ahead of polyphosphate synthesis. This fact would support the hypothesis, discussed earlier (6: 20: 31), that polyphosphate is synthesized on the surface of RNA and then both are utilized. Whether the chloramphenicol induced delay in RNA loss after 9 hours of light was due to synthesis of RNA with different properties or to the specific utilization of polyphosphate but not RNA, is not known. Takeda (132) reported that the RNA,which was synthesized in the presence of chloramphenicol, exhibited different physical properties than normal RNA. In the dark part of the life cycle a rapid build-up of RNA occurred in the first six hours with very little increase in polyphosphate. This was then reversed in the last six hours of darkness. If the lights were left on for three hours longer than usual, this divergence was not noted. This effect of light on RNA synthesis was already noted in Figure 15 in the work with Anabaena. Further evidence that light influences the quantity and nature of RNA, independent of the life cycle, is shown in Figures 29 and 30. These algae were not synchronized. One culture from.which the RNA-polyphosphate was isolated had been exposed to a high level of continuous light and almost all of the RNA eluted at a low salt concentration, (Figure 29). In another case the algae were grown under a low level of continuous light and the RNA eluted at a higher salt concentration,(Figure 30). In the operation of the Chlorella synchronization 116 Figure 29. Elution pattern of total RNA and phosphorus from a random culture of Chlorella grown under continuous high light intensity (solid line = ug. total-P/ml./10 m1. p.c.v.; dotted line 3 optical density at 260 mu/lO ml. p.c.v.; salt gradient in this case was the same as normally used for 52222223 elutions.) 117 ' '[Iu/g-t'sqoq °Sn 0m oasta .3 Quad 035 0a 00 0m 0: 0m 0« 0H H q - 4““1“'1‘ - d\....l. COO OOOOOCO.|\AQOOOOOII .. ‘0'... 010-..-.‘0\-I| L H00 .1 N.o 1 m.o L +70 1 m.o 1 o.o 1 To I $00 1 0.0 J 02—” 1 H2... 'CI '0 mu 098 4'3 118 Figure 30. Elution pattern of total RNA and phosphorus from a random Chlorella culture grown under continuous low light intensity (solid line- ug. total-P/ml./10 ml. p.c.v.; dotted line - optical density at 260 mu/lo ml. p.c.v.) 0m mastm .3855 can» 00H 00 00 0e 00 0m 0: 0m 0« 0H H - 1 H H . q H a H We 0.0 eOeQeoeb eed OKe-ejet 0‘ I H e O |e§0§eeet .— IO. 0‘ O l N03 x .. .. a. O N .— 0 I. O a.” K ~ m 3 .. ... n . 1 1.0 .. a. ._ . .. .. . . .. 3. w .\ _ ._ 1 0.0 . L. _ x a. \II fl ‘ I. NCO _. .0. . \ . . J 0 0 . .0 1 0.0 .. .. 1m. 1.... 3 . a r 1. 3 ‘!e he 0.. 0 run 092 4'9 ’(I ‘0 120 apparatus it was noted that a culture had to be quite dense to withstand continuous high illumination. In several cases the use of high illumination too early in the establishment of a culture resulted in clumping and loss of the culture. Part of the cause of the observed effect of high light on RNA synthesis might be photo-oxidation. Sager (11h) and Anderson (5) have obtained evidence that plants deficient in carotenoids can not withstand prolonged high illumination due to the photo-oxidation of their chlorophyll. Anderson suggested that carotenoids might also protect such porphyrin- containing substances as catalase and the cytochromes. In Table XII the amounts of total RNA and total phosphorus are recorded and summarized from the fractionations illustrat- ed in Figures 8-lh, 16-27, 29 and 30. It is interesting to note that the ratio of total RNA to total phosphorus in the middle part of the Anabaena growth.curve was about the same as in synchronized Chlorella in the light phase of their life cycle. The very low ratios observed in continuous high light are compatible with the fact that no shortage of ATP would have existed under these conditions and, consequently, an accumulation of polyphosphate could occur. Changes'Within RNA-Fglyphosphate Areas During the Life Cycle Figures 31 and 32 illustrate the changes in amount of RNA in areas A-F during the life cycle. Figures 33 and 3h give the parallel changes in percent distribution of RNA among these areas. 121 All of the areas reached a peak magnitude at the nine hour light stage and areas B, D, and E also reached a second peak at the six hour dark stage. Area F, which required the highest salt concentration for elution, showed the greatest changes. The main effect of chloramphenicol was a large increase in areas B, D, and E with almost no effect upon the rapid utilization of area F. Changes in the magnitude of the phosphorus areas are shown in Figures 35 and 36. Changes in percent distribution of these areas are shown in Figures 37 and 38. It is inter- esting to note, that at the six and nine hour light stages IV and V'were the dominant phosphorus areas, whereas in the case of RNA area F was dominant. It is possible that as the RNA of area F became phosphorylated it was eluted at a lower ionic strength and appeared in areas D and E. Such a hypo- thesis is supported by the fact that area F of the RNA reached its greatest percent of the total RNA at the six hour light stage although there was slightly more area P RNA at the nine hour light stage. Areas roughly corresponding to the six RNA and poly- phosphate areas were pooled and analyzed for total-phosphorus, A7-phosphorus, and metachromasy. Aliquots were then dialyzed 2h hours against distilled water and analyzed in the same manner. Ultraviolet spectra were taken of both the dialyzed and non-dialyzed samples. With these data an idea of the dialysis rates and relative amounts of undenatured complex can be obtained. In Figure 39 the specific metachromasy of 122 Table XII. Influence of light upon the ratio of total RNA 0. D. units to mgm. total phosphorus (one RNA 0. D. unit = one ml. of a solution with an O. D. @ 260 mu.of 1.0) ' RNA units/10 mgm. tot.-P/ RNA unit Fige organism conditions ml. pQCOv. 10 mle P.C.V.»mgm.TO'E.- 8 Anabaena 3.6 ml. 13.9 1.82 7.7 p.c.v./1. 9 " 7.2 m1. 10.1 0.51 20.u peceVe/le 110 " 8.0 ml. 10.3 0.59 17.6 p.C.V./1. 11 " 11.2 ml. 5.1 0.31 16.5 p.c.v./l. 12 " 13.0 ml. h.6 0.23 20.u p.c.v./1. 13 " 25.0 ml. 3.0 0.10 21.0 p.c.v./1. 1h " 29.6 ml. 7.7 0.13 58.0 A p.c.v./1. 16 Chlorella synchron. 75.3 3.29 22.9 17 " synchron. 98.0 5.11 19.2 3 hr.light 18 " synchron. 172.3 6.32 27.3 6 hr.light 19 " synchron. 220.5 11.12 19.8 9 hr.light 20 " synchron. 59.9 3.25 18.h 12 hr.light 21 fl synChrOn. 9700 “.082 20s]. 22 " synchron. 7l.h 3.73 19.2 18 hr.light 26 " synchron. 99.0 5.32 18.6 21 hr.light 23 " synchron. 109.3 3.95 27.7 3 hr.dark 2h " synchron. 163.2 h.9h 33.0 6 hr.dark 25 " synchron. 82.5 3.05. 27.0 9 bredark 27 " synchron. + 177.0 h.h8 39.5 chloramphen- 1001 29 " random,cont 37.6 3.96 9.5 high light 0 " random,cont 268.0 7.89 3h.0 low light 123 ApanH 0H manon Hm n 4 .panH QH memo: m as @0000 HooHGonmEmaodnonun.onHH UHHom u c.0090 .onHH noxoan u m «one .ocHH coupon u ¢ mommy nnzoaw afloQonnoqhm wdHhsv H H383 npzohw neocohnochm Madge opdfimmonfinHon dHHohoaflu HHH newness .om cadmfim fillxhwp 5H canon L. paw: 5” unnosllli H m o m H H NH o b m o . l- - \ I/ \O\\ / J . v o C O C C N H 0 e M 'A'O'd "[111 OI/J-Isqoq °u13ux 130 Ava—ms...” HS” madam Hm u 4 .pnmfl” a mason o no poops Hoodaohmamnoano n D .2»: paaom u HHH dead .23..” demons. H mm dead .03..” @9306 ..I. H «083 npzohm 9938.503? warned encammonfinaom 9:93.30 a.“ sewage .mm onstm TII-thp 5” agent's—o paw: a.“ mason ma 0 o m. ma H NH 0 o m H . J t H H d 5‘... ....O. ...... D s... ....... ........ .. .. . \vi/ 3.0... e .3... \R5 ...~-\ // \Oll‘l . /...... . . u ...\ . .... \ (\\ . us ..I [0“ Q ..... ... \ .. .. at. .. H ...“O.....\ D \ .uwzgzaxxua. .r .\ \ elu.|. . .K 0H 10m is 5-13404 30 g 131 AphmHH fl 982 Hm u 4 .pstH 5 assoc o as cones HooHcofisssloo u 6 JG: .033 n H> sons .23." genome. u > moss .03.... popped n .5 scam; npzonw mnohopdofihu marge epmflmmongaom 9:93.30 5 homage .wm 0.3th Tl asap 3.. wagon nu.» NH 0 \ ......... .....,......... J / - .. \ x D. .. // \OJ ... .....M A .. Vi.!......¢.\ ./ ..Q.......\\\.. /.N...0\ : \ ..H ~ 0.. .. .\.\ \ f/ L .. .. ...... I.. . .O. uthH sH usaohllli . H m. o n o ON on o: 13404 J0 % a- 132 ApthH 5 Push Hm u 4 :EmHH sH mason m 2. H533 HooHsohmsa 33?? n D .093” peppovunoxono. u .m ache .2”: Samoa n w some .083” demons. u a some 5:: coppoc u 0 dead? head: ceHHHpmHo 9353mm aamhaaao monks uoKoHnSoo opgmuonthomu4zm saaohoaflo no humaonnocpoa oHHHoomu .me mgm.—”m +t||||||.xsse sH assos.ulnnul4n uanH sH «wean .— .sl. .. t . A ...0... \& 0......‘31...... . \ x .; ..... .gqxaxa. x mm ”u .xvx. o.o m.o 4.0 0.0 w.o 0.0 0.4“ .mH o.N “00.: 0.0 05 o.oH 0.:H o.mH fissmoaqoeqem outoeds 133 changes may be the reflection of the hyperchromic effects of dialysis, particularly for those points that show negative losses. They may also be a reflection of the degree of de- naturation rather than the molecular size as will be demon- strated in a later section. Although care was taken in fill- ing and draining the dialysis tubing, a small amount of dilution undoubtedly took place in the process. It is inter- esting to note, however, that some dialysis of RNA took place at some stage of the life cycle in each area with the possible exception of area F. Likewise some hyperchromicity was also demonstrated for all areas except D and perhaps F. If the maximum change during dialysis is noted for each area, the percent of dialysis will be found to decrease from area A to area F in sequence. This is in agreement with the literature reports that the molarity of elution of the nucleic acids are roughly correlated with their molecular size in modified-cellu- lose ion-exchange chromatography (12, 1314). In Figures h} and hh the percent loss in acid-labile phosphorus upon dialysis is given for the six areas during the life cycle. Some omissions were due to an insufficient amount of polyphosphate to carry out accurate experiments. One of the interesting facets of these data is the fact that, although neither area A nor area B ever showed significant l“'le‘lsachromasy before or after dialysis, they never lost all of their A7 -phosphorus upon dialysis and at times they did not 1°8e any. These facts are contradictory at first sight, 31.an short polyphosphate chains dialyze completely 13h SthH 5 Push Hm u 4 ...EwHH sH Econ o as cases H8339... 3.3.30 an .033 ooppoounoxoao. n .m s93 .oHHHH 0.30m n m sons .oHHHH H333. n a some .09: ooppoo u 0 H.930 .Hmwoz ooHHHpmHo page? namhasao moms 383.930 opgmuongaomiwzm «Haemogo .3 hpamgoanompofinonhn .0: ongm Tense 5 3:34 33H fl 953 .H_ H o . m H mH «H a o m u 4 : u - J .. 4 . . o I - 0H mull \‘O \ a 1 0m l 10\ ...m.. /// \ I .. on \ a. ...... /” \\ I \ .. .. W4 ’ - 3 i .... . .. , ... . a \ ...... . . . [a w . 1 8 \ .. . .... . .. \rh .. a \\ \ \‘ . U E. / f,’ of ' ck 8 53 Ionlll. _ OOH EU: on em 'oeds ssmoaqosgem 013109 8 v Esswo 001: X 135 ”Em: 5 maxi S u a Sam: 3 mason o 3 323 303395320 u D mafia vHHom n 0 «ohm .onaa doxonp a mucous .ouHa weaned n « mogmv Mona: noaaapmdv pmnawmd mamhadao dean «am wHHQAOHno no mmOH .H: ohawam T 33% fi 2:0an 13m: 5 mason a_ ma o 4 o m 3 ma ma 9 o m d '-m on: u d .88...va .... .. om- O H I °q '0 am 092 JO ssot % 136 magma” 5 mason Hm n 4 Jam: 5 «.80: a em. 323 303333030 n a 25H Eamon n m 8.3 .033” demons. n m no.3 .051” @0306 u n dong nova: vaHHumfin pmnawwd mdmhawac moan 3E 93090.30 .Ho mmoa .N: 93me HIIIMES 5 masonlfi fimfi 5 was: .— H o o m H ma 0 w4 d . a q - ma 0 o m . 1 W 4 Om..- 1 cm... 1 0...... dmr 'q ’0 “In 092 JO 8801: 137 under these conditions (23) and long chains give a strong mmtschromatic reaction. The most logical solution to this problem, it seems, is to assume that the polyphosphate was in the form of short chains, which give no metachromatic reaction, and that these chains were covalently bonded to RNA so that they would not dialyze. No previous reports of such complexes involving low molecular weight polyphosphates exist. Some Preperties 3; Various Isolated Fractions gg’RNA-Polyphogphate \ changes in chromatographic pgtterns 2£22£ dialysis Figure hS shows the elution pattern obtained when a sample of the complexes which elute at low ionic strength was dialyzed and then rechromatOgraphed on a DEAE-cellulose column. The peaks were not shifted very far and, although there was a loss of 30 percent of the total phosphorus and no percent of the 260 mu 0. D. upon dialysis, the distribup tion of ATphosphorus and RNA-phosphorus was about the same in the pooled areas of the original column (Figure 29) and the column in fligure us. Other than the losses due to dialysis there seemed to be little effect. . An aliquot of the pooled complex from the original column (Figure 29) was tested for an increase in metachromasy after freezing, five minutes of boiling, or'60 minutes of incubation with RNase. Although a very small amount of meta- chromasy could be detected in the original complex, no 1 “"3 flu-s. (-5 “I, ‘ ’9‘ . 138 haul-ll» _ w [Ibl‘bfil I .. . t n. .... . Aw I 3mm: 3 mason am u e 3%: 5 mas: o as cocoa 303333030 nu gonad uaaoo u u some .onaa noxonn u m «one .ommH weapon u 4 woaov mono: voaaduuao pmnaomm odmhasdo cogs oponmnonmhaom saaonoano we need .m: onzwam ”Illness 5 fiscal ..Ewfi 5 mason L— o 4w" i! mmw fl. m mm mm NW mm ‘M: aux smoudsoqd-Lv 50 88013 g", 139 3%: 3 982 .8 u 4 3%.: 5 mason m em 333 flooagoflafiofio n a 6:: oaaom u .m wand .99: anemone. u m wold .oaHH wompoc u n .383 noun: ug.—H.330 pmnawwa mauhawau moms ouwnmaongaom waaonoanu no mac." ....3 ogmum filafiu 5 38ml» 33H 5 was: NH 9 0 m 0H mH NH 14 . . _ q l x. ......N....... or... F... \O... .......0 ”......ux... ; .... .. O“... 0 O .\. ....p: sly»: . 0. Oz 0....\ \ \ // \7 I In\\ 4— WW0 ..OH O N l l I o 53 3x 3 m snaoquoqd-Lv JG 9901; % I O (s. 1&0 increases could be detected after these treatments. When area E complex of the relatively undenatured type characteristic of the six and nine hour light stages was dialyzed and rechromatographed on DEAE-cellulose, a consider- able change was found. Figure ho illustrates the new elution pattern and the centers of the original areas occupied by the pooled RNA and polyphosphate are indicated at the tOp of the figure. A new small peak of free RNA was located at about tube 12. The major changes were a shift of the centers of RNA and.polyphosphate to a position 16 tubes earlier in the elution and a considerable sharpening of the "peaks.” The fact that the RNA and polyphosphate peaks do not cor- respond in Figure ho suggests that the material was not homogeneous and that the complexes which eluted first had a higher percentage of polyphosphate. Metachromasy was tested on tubes h8, 56, and 62. All three had significant values. The shift in elution position of the polyphosphate in Figure ho after dialysis suggests that the polyphosphate was connected with RNA either before dialysis, after dialysis or both, since free polyphosphate should have been uneffected by dialysis. 23.9.2 ratios An effort was made to determine base ratios in the different elution areas as an independent verification that the various RNA peaks were actually different and not merely polymers of each other. Table XIII gives the base ratios _ 35;}. If.“ Aaowz z N on o u pqoaoonw wade “nope: ooaaaamao puqadwa mHthaMm nevus 0N .wah Song om nmdonnp 0a mops» penanaoo mo noaunom av wHHohoHno_anH mowoamsoo unmaoz nodsooaos 30H oouhawwo no obnSo noapfiao dzm .m: ohswam lhl honasn can» N 0 Oh 0a . OT OM «m OW J wN a H o O 0000000000000000000') 0000000 00000000 0» 0000.0 N I. H00 . in \J ~ . N L 1 m6 a a». _ pu— _ 1 m.o . . — _ _ _. .. :5 _ q “u L m5 —. fig .1 000 L To oxwom unnonmmonm awuop was lam 3909 no whopnoo Handmano nm 092 49 'Q '0 “nope: uoaaapmae p.caama mamaaaae sebum ma was we .mmHa 80.5 w some Molcoaunom 3 hope: ooaaapmac pmfiwwo mdmhawac .3pr KonSoo m mono «HHonoano .Ho admonmmonm one 58 Hmpon no 589me £0,336 .0: mgm.—”m hogan cob» 0.0 N.o n .z o a 1. ©.o o 2 .... m... Q6 & w . o..." ONH .. N...” 3 . Ofi I. — — I. 30H . _ u h 00.7. ohowoo. hopMMOHNWW lilL — . unopnoo 30a 1 0...” Monaco agonhmonm Hodfimaho nm 092 4‘3 'CI '0 1&3 found in the RNA areas from the nine hour light stage. This stage was ideal for this type experiment since all six areas were clearly present at the same time. It was not determined whether the base ratio was the same in these areas at other stages of the life cycle. It is obvious that the base ratios were different in the various areas at this stage of the life cycle. It would be interesting to determine the base ratios at other stages of the life cycle and perhaps, thereby construct some interconversion sequences. Similarly it would be interesting, but laborious, to follow the uptake of P32-phosphate into the various complexes in this system. ribose and deoxyribose The pooled areas from the randomly grown Chlorella, (Figure 30), were each analyzed for ribose and deoxyribose. No deoxyribose could be found. The ribose values are given in Table XIV. Calculations were made of the ug. ribose per RNA 0. D. unit in areas D and EL These came out 12.9 and. 16.9 ug. respectively, and a value of 15 ug. ribose per RNA 0. D. unit was arbitrarily taken for approximate calculations. The value of 20 RNA 0. D. units per mgm. total-phosphorus has been derived (Table XII). It can then be calculated that for every micromole of ribose in the total system of complexes there were about 16 micromoles of phosphorus of which 15 micromoles would be polyphosphate-phosphorus. This value was about the same for Anabaena in the middle part of mu its growth curve and for synchronized Chlorella in the light phase of the life cycle. Further Characterization 2£.§ES Complexes Figure h? illustrates the infrared spectrum of material from area D of the nine hour light stage, before and after dialysis against distilled water. The band at 7.7 to 8.0 u is characteristic of linear polyphosphates (26). The only qualitative changes evident upon dialysis were in the region from 12.2 to 13.0 u. A small peak appeared at about 12.6 u in the dialyzed material. This region is characteristic of the molecule as a whole rather than of its bonds. The area E complex from the random Chlorella shown in Figure 30 was used to study the effects of a number of treatments on its metachromasy. The results are given in Table XV. Since dialysis against distilled water seemed to be the most effective method of releasing metachromasy, a study was carried out to see what was responsible for this change. Two possibilities seemed likely. One was the possibility that the pH of the distilled water was low enough to cause a selective hydrolysis and the other possibility was that the low ionic strength, which is known to cause the cleavage of such hydrogen-bond systems as involved in the DNA double helix, might be causing the change. By dialyzing samples of areas D and E against buffers of known pH and NaCl con- centration these two possibilities were tested. Figure h8 1&5 Table XIII. Base composition of the RNA in.the pooled areas from the nine hour light stage of Chlorella .molEpercent area adenine guanine 0MP UMP A 35.3 1h.1 10.8 39.9 B h5.6 22.h 7.9 2h.1 C 18.3 27.8 9.9 hh.0 D 26.8 32.9 16.8 23.6 E 26.7 25.9 16.1 31.3 F 3u.2 19.8 20.8 25.2 Table XIV. Concentration of ribose in the pooled areas from Figure 30 (random.Chlorella) area ribose (ug./ml.)} h.0 22.2 19.9 8.3 1h.7 63.2 WHUOU’» 111,6 Ahopmz poaaapmao umcammm mamhamao songs 550on o>apmpaa§u u 053 doggone; 9:93.30 oouanohnonhm .ommum unmaa melon o .9 menu ho glaucoma oonmmnuqa .u: onfimHm ed 5 NH onoaofia 3 OH w : 11 1 q‘ s u . ~ ~ ~ s D. A a \ o d :13“ OH O N o o o o ~o In ;: cw ectrsqnnstmaq % O p cm 1&7 shows the effect of pH at two concentrations of NaCl, and Figure h9 shows the effect of NaCl molarity at pH 8.0. The specific metachromasies are accurate, since ATphosphorus was determined on each sample after the experiment. There seemed to be a pH effect at a low salt molarity, but not in 0.1 M NaCl. It was fortunate that the pH used in both the phenol extraction and the DEAE-cellulose chroma- 7..— tography was in the pH region of greatest stability. This complex was apparently stabile in NaCl solutions above 0.2 M if the pH was between 6 and 8. In Figure h9 the percent loss of A7-phosphorus is also plotted. It was inversely related to the amount of denaturation of the complex. The interpretation of these data could be that the dialysis denatured some tertiary structural system in the RNA, resulting in hyperchromicity and exposure of the polyphos- phate. The fact that boiling and the alkaline pH utilized in the isolation failed to release metachromasy argues against this interpretation. Another possibility is that the polyphosphate chains were involved in a hydrogen-bond or a salt-bridge system.with the nitrOgen bases of the RNA at intervals along the polyphosphate chain. Such a system might exhibit the observed properties. Effects 3; Adsorption gg Charcoal On the basis of a charcoal separation procedure, Lisa (89) believed that polyphosphate and RNA were separate entié ties. When charcoal, prepared by the method of Lisa, was 1h8 Table XV. Effects of various treatments on the metachromasy of area E RNA-polyphosphate from random Chlorella (Fig. 30) specific ' treatment metachromasy none 0.07 2h hrs. dial. against distilled water 1.7 freezing and thawing 0.07 10 min. boiling 0.0h 12 hrs. treatment with.Na'EDTA 0.07 18 hrs. dial. against 0.01 M acetate, pH 5.5 at 2° Ge 10“- 18 hrs. dial. against 0.01 M acetate, pH 6.0 at 20 Ce 102 1&9 Figure 148. Changes in RNA-polyphosphate upon 10 hours dialysis at room temperature (Solid line: 0 hour light "sample, area D; specific metachromasy before dial. was 0.07 and after dial. was 1.50; dialyzed against 1 l. 0.01 M tris or acetate buffer. Broken lines 3 hour light sample, area E; specific metachromasy before dial. was 0.09 and after dial. was 1.27; dialyzed against 1 l. 0.01 M tris or acetate buffer with 0.1 M NaCl added.) 150 b 0.8 k 1.6 1.5 1.0 1.3 1.2 1.1 1.0 0.9 0.6 0.0 0.3 0.2 0.1 hmdaonnompos ounaoomm .0 7. 7.0 pH of buffer °°° 6.0 Figure h8 151 Figure 09. Changes in RNA-polyphosphate upon dialysis for 16 hours at room temperature against one liter of 0.01 M buffer, pH 8.0 (6 hour light sample, area D; specific metachromasy before dial. was 0.025) 152 A093“ demons; mLé Ho 039300 R 0 0 O 0 0 0 0 O 0 0 O 9 8 7.. 6 5 h. 3 m 1 1 p o. q q — _ .Afl. J a a 2 O 0 I J / l / . 5 10 v 0 L 0 p 0 0 0 9 3 7. 6 S h. 3 2 1 0 e 0 0 0 e e 0 0 e 0 e 1 O 0 0 0 0 0 0 9 O 0 A093” p.303 hmgofinocuoa camaocmm molarity of NaCl Figure #9 153 used to attempt the separation of the polyphosphate and the RNA in various complexes the results of Lies could not be duplicated. When ribose was determined before and after adsorption it was found that all but traces of ribose were removed. However, relatively large amounts of A7- phosphorus were sometimes also removed. This loss varied from six to 1414 percent of the A7-phosphorus. Erratic, but large, effects upon the specific meta- $81 chromasy of the polyphosphate remaining after charcoal ‘ - adsorption were also noted. In order to resolve this prob- lem two experiments were run. In one, samples of complex J from various areas B and D were used, and the results are .fizt? shown in Table XVI. In the other, synthetic polyphosphate was used and the results are shown in Table XVII. Because the charcoal destroyed the metachromasy, it was apparently catalyzing a slow and probably random cleavage of the free polyphosphate chains. Also a significant percent cf the free polyphosphate was adsorbed by the charcoal, thus making any attempts to separate naturally occurring RNA-polyphosphate with charcoal of doubtful value. These facts are important in the interpretation of the data in Table XVI. The data on area D are the most important. The fact that A7- phosphorus was released by the action of charcoal and was then able to dialyze, even though the samples had been dialyzed previously, indicates that the charcoal also catalyzed the cleavage of the polyphosphate in naturally occurring material. This was also indicated by the fact that 15h the specific metachromasy of the charcoal treated complexes after dialysis was still below that of the dialyzed complex which.hadn't been treated with charcoal. In the case of the three and six hour dark samples of area.D, (Figures 23 and 2h), no specific metachromasy changes were obtained. These samples also showed very little increase in specific meta- chromasy when dialyzed (Figure 00). Thus they were probably composed primarily of free polyphosphate of considerable F length. Quite a few random.cleavages of a long polymer would be needed to lower the specific metachromasy very much. Hyperchromicity g; RNA-Polyphosphate Some experiments were carried out to determine whether a difference could be detected between the hyperchromicity of native complexes and denatured complexes, when they were boiled and then cooled rapidly;(Table XVIII). Areas D and E from the 6 hour dark Chlorella showed very little hyper- chromicity. This corresponds with the fact that these areas showed very little increased metachromasy upon dialysis, (Figure 00). In the case of areas A, C, and D from.F1gure 27, significant amounts of hyperchromicity were observed. Dia- lyzed aliquots of these areas showed consistently lower but still significant amounts of hyperchromicity. Enzymatic Studies characteristics 2; the yeast pglzphosphatase complex This enzyme complex was remarkably stabile at room tem- perature and seemed to be made up mostly of pyr0phosphatase 155 . . om.H mm.a ma masons .em .waa . . FH.H :H.H we a «on. .mm .waa em.o a: om.o 0H.H an Hoaz u H + a son. .Hsae .pm .maa eo.o mm mH.o :a.o me a some .am .maa oo.o we oo.o oo.o Hm scan a H + m «on. .Hsae .am .maa 00.0 o: oo.o eo.o aw m «on. .hm .waa humaohnosuoe mafia Hsoego Hooogo mL. 6 oauaoomm Ho mmoH hound enouoo mood m Heanopda m Hana» hmdaoanOdpoS .oono nuclease» deconsne nope. maasaaae ndmhaoap ohohon escapees» Heoonmno osqsaaosaaaoaudzm.saaoaoasu no open. m muons powhaoapns one powhasup some oduhadap one Hooondno Ho upoohmm .H>N edema 156 Table XVII. The effects of charcoal upon synthetic polyphosphate in a one percent NaCl solution percent loss of 57-13 35.0 specific metachromasy before treatment 1 .28 specific metachromasy after treatment 0.26 44% A ‘ 157 and a highmolecular weight random.polyphosphatase. That other polyphosphatases existed in the complex was illustrated by the activity on tripolyphosphate, but this activity was relatively low. ‘When measured by orthophosphate release the action of the enzyme complex was very slow on synthetic polymers with an average length of 16 phosphates and not very much faster on tripolyphosphate, (Table XIX). This low activity was not increased when the enzyme was added in portions during the incubation and the activity was inhibited by orthOphosphate. The fact that a very active, random cleavage polyphosphatase was present is clear, when the data in Table XX are considered. Decrease in metachromasy is a function of polymer length and three to six minutes was sufficient to reduce the metachromasy of‘the synthetic polyphosphate to 30 percent of the original level. This enzyme complex had no activity against AMP or the triphos- phate chain in ATP. action g; yeast polyphosphatase gg_RNA:polyphosphate When the same yeast polyphosphatase preparation was incubated with RNA-polyphosphate from area D of Figure 27 for ten minutes, the specific metachromasy decreased from 0.1h to 0.09. Then an aliquot of the enzyme treated material was dialyzed against distilled water for Zh hours. A de- crease of 23 percent in Luz-phosphorus was observed and the specific metachromasy increased to 0.87. These values are to be compared to a loss of only four percent of the A7- phosphorus and a specific metachromasy of 1.16 upon dialysis " . . a. ‘ 158 Table XVIII. The hyperchromic effects of boiling Chlorella RNA-polyphosphate for ten minutes, followed by rapid cooling “—2280Vmu 0. D. sample before A after A hypegglziggrmicity 6 hr. dark, area D 0.37? 0.380 0.8 6 hr. dark, area E 0.506 0.516 2.0 Fig. 27, area A 0.127 0.1M? 15.8 Fig. 27, dial. area A 0.236 0.252 6.8 Fig. 27, area C 0.237 0.262 10.5 Fig. 27, dial. area 0 0.236 0.253 7.2 Fig. 27, area D 0.673 0.759 12.8 Fig. 27, dial. area D 0.639 0.678 6.1 159 Table XIX. Release of orthophosphate by the action of the yeast polyphosphatase complex on known substrates assay: 2 ml. substrate, 0.1 ml. of 0.015 M MgCl , and 0.0 ml. of enzyme preparation; incu ate at 30° 0. Final concentrations of total-P and ortho-P were determined; all substrates adjusted to pH 7.6. ncubation 3g. ortho-P/ml. substrate time . nus endogenous ug. total-P/ml pyro-P 30 min. h26 770 tripoly-P 30 min. ?h 1100 10 hours 297 " " 20 hours 1.00 " " 6 hours h§0 " " 0 hours 1. o " synthetic pgly-P l min. EB 1025 5 min. '1 It 15 min. “'5 It " " 30 min. 52 " n n 1 hour 51+ to II It 1 hour * 7 ll " " 10 hours 0 " " " 20 hours 95 " " " 6 hours 105 " " " 0 hours 1 0 " " " 8h hours 0 " synthetic poly-P ** 30 min. 0 560 ATP 30 min. 0 550 AMP 30 min. 1 650 * 0.1 m1. enzyme added at 0, 15, 30, and #5 minutes ** 50 ug. ortho-P/ml. added to substrate before the enzyme 160 of untreated area D. When the enzyme-treated and dialyzed area D sample was then subjected to polyphosphatase action again, the specific metachromasy declined rapidly to 0.37 after three minutes and 0.31 after six minutes. This indicates that the random cleavage polyphosphatases had low activity on "native" RNA-polyphosphate, but were effective on dialyzed RNA-polyphosphate. In the latter case the polyphosphate chains would have been released from their hydrogen-bonding to RNA and wouli have been subject to the attack of the enzyme. __.__act1°n 2.1: 819.22 292 111.332 .2922 phosphodiesterase 2g,RNA-polyphosphate As discussed in previous sections RNase failed to re- lease the metachromatic reaction of undialyzed RNA-polyphos- phate complexes and in some cases decreased the metachromasy. It was also observed that neither RNase, isolated snake venom: phosphodiesterase, nor 1y0philized snake venom had any effect on the metachromasy of synthetic polyphosphate, thus eliminat- ing the possibility that they contain polyphosphatases. These enzyme preparations were then tested on several RNA- polyphosphate complexes, (Table XXI). In the case of dialyzed area D from Figure 27, after it was incubated with RNase for 20 minutes, and then with snake venom phosphodiesterase for another 20 minutes, an aliquot was dialyzed. After dialysis the specific metachromasy was 0.38 and there was only about a 15 percent loss of A7-phosphorus. Areas D and E from a random Chlorella.culture were tested in the same way except 161 Table XX. Effects of yeast polyphosphatase on the metachromasy of synthetic polyphosphate assay: 2 m1. substrate, adjusted to pH 7.6, plus 0.2 m1. enzyme preparation; incubated at room.temperature minutes metachromasy specific incubation (ug. hexameta-P/ml.) metachromasy 0 13.9 1.0 3 5.1 0.37 6 h.2 0.30 162 that a solution of 1y0phylized rattlesnake venom was used instead of purified snake venom phosphodiesterase. In this case very little effect on the metachromasy of area D was found, but the metachromasy of area E was found to be effected in about the same way as area D, Figure 27. These effects of RNase and snake venom phosphodiesterase on the metachromasy of RNA-polyphosphates are another piece of evidence that a connection exists between the RNA and the polyphosphate. Since after the action of the RNase and snake venom phosphodiesterase or the action of polyphos- phatase, dialysis fails to remove very much of the polyphos- phate, it seems likely that the polyphosphate chains are covalently bonded to the RNA. One way in which this could occur would be by means of a phosphate ester involving the secondary hydroxyl at the end of the polyphosphate chain and a hydroxyl of the ribose in RNA. Such a structure would be in accord with the observed properties of RNA-polyphosphate complexes. It would account for the very high specific metachromasy observed in dialyzed complexes since the secondary end hydroxyl is inhibitory to the metachromatic reaction. The effects of RNase and snake venom phosphodi- esterase could be due to removal of RNA obscuring the surface of the polyphosphate or to cleavage of the RNA chain in such a way that it was more random in arrangement and thus inter- fered with the metachromatic reaction on the surface of the polyphosphate. Another possibility is that the snake venom phospho- 163 Table XXI. Effects of RNase and snake venom phospho- diesterase on the metachromasy of Chlorella RNA-polyphosphate assay: 2 m1. RNA-polyphosphate solution, : a few crystals of RNase and a 20 min. incubation, i 0.2 ml. snake venom phosphodiesterase for a variable number of additional minutes of incubation IRN snake venom incub . specific substrate ase P-diesterase time ) metachromasy 6 hr. dark, area 0 - - 0 0.00 6 hr. dark, area 0 + + 20 0.00 area D, Fig. 27 - - 0 0.1a area D, Fig. 27 + + 20 0.00 dial. area D, Fig. 27 - - 0 1.16 I! II N + + 20 0 e 2 n n w _ + 5 0. 8 " " " - + 10 0.8h n n N _ + 20 0.76 n u H + + o o .91} " " " + + 5 0.82 " " " + + 10 0.75 " " " + + 20 0.33 dial. area D, from - - 0 0.63 a random culture . " + + 0 0.55 " + + 5 0.66 " + + 10 0.61 " + + 20 0.66 area D, from a random culture - - 0 0.19 " + + 2 0.2 area E, from a ‘ random culture - - 0 0.56 1 " + + 20 0.39 dial. area E, from a random culture - - 0 3.1 + + 0 1.6 N + + 5 1 e 1 " + + 10 1.2 “ + + an 1.1 16h diesterase cleaved an ester bond between the polyphosphate and the 5' hydroxyl of ribose in the RNA. Such activity is possible, since Razzell (109) found that this enzyme attacks ATP slowly, releasing pyrophosphate, but not orthophosphate. It also attacks di(thymidine-5')-pyrophosphate to yield thymidylic acid at a rate ten times faster than it attacks PTPT. In a similar manner RNase might hydrolyze an ester between a pyrimidine 3'-hydroxy1 and the terminal secondary hydroxyl of a polyphosphate chain, since it is also a phos- phodiesterase with.specificity only for the pyrimidine 3'- phosphate portion. An example of this type of activity is the action of RNase on the synthetic methyl ester of 3' UMP as reported by Brown (17). No matter what the mechanism of the effects of RNase and snake venom.phosphodiesterase on the metachromasy, the possibility exists that some or all of the polyphosphate: involved in the complexes was relatively short in length and gave a high specific metachromasy after dialysis by virtue of the fact that the two ends were esterified to RNA. This possibility is supported by the fact that mild alkaline hydrolysis, which does not hydrolyze polyphosphates, drastic- ally reduces the specific metachromasy of RNA polyphosphates, (Table V). It is also supported by the fact that yeast polyphosphatase reduced the metachromasy of the complex rapidly, but did not release the polyphosphate to dialyze. For example, the metachromasy of dialyzed area.D, from 165 Figure 27 was reduced to 17 percent of its original value within five minutes by the action of yeast polyphosphatase. CONCLUDING-REMARKS In this section an attempt will be made to postulate an overall picture of polyphosphate metabolism. This picture will be based upon the author's knowledge of the literature and an extrapolation of the data presented in this thesis. As discussed earlier many microorganisms incorporate orthophosphate into high-molecular-weight, organically bound polyphosphate very rapidly. This incorporation apparently takes place by way of ATP. The result is a complex which involves RNA, polyphosphate, and probably other components as well. This incorporation of orthophosphate into polyphos- phate only occurs at one stage of the cell-division cycle. The resulting RNA-polyphosphate has a very high molecular weight and gives no metachromatic reaction. It seems likely that the RNAportion of this complex is synthesized first, and that the polyphosphate is then elaborated by an enzymatic process involving ATP. As this complex is phosphorylated its point of elution from DEAE-cellulose is moved to a lower ionic strength, probably due to changes in the folding and number of exposed charges on the molecule. The structure, which the author considers most likely to be present in this newly synthesized RNA-polyphosphate complex, is one in which polyphosphate chains of various 166 167 lengths form bridges between the ribose mdpties within RNA chains. Such bridges could be terminated by an ester bond at each end. This ester bond could be formed by splitting out water between a ribose hydroxyl and the secondary hy- droxyl of the terminal phosphate of a polyphosphate chain. These polyphosphate bridges might connect separate RNA chains or form links between nucleotides on the same RNA chain. In the original “undenatured" RNA-polyphosphate complex these polyphosphate bridges seem to be involved in a hydrogen- bond system with the RNA. This system could be composed of a series of hydrogen-bonds between the phosphoryl-axygens in the polyphosphate chain and either the amino nitrogens of guanine and cytosine or the nitrogens in the purine and pyrimidine ring systems of the RNA nitrOgen bases. Such a hydrogen-bond system might have the unusually stabile prop- erties observed in the isolated RNA-polyphosphate complexes. No reports have been published which demonstrated a definite metabolic function of the RNA-polyphosphates. The author feels that the most probable metabolic function of the RNA-polyphosphates is protein synthesis. This function has been indicated by several factors. As was discussed earlier, the physiology of the volutin granule plastids in several microorganisms points to their utilization in energy-requiring processes at sites of active growth. Also the kinetics of high-molecular-weight polyphosphate utilization in unicellular organisms has 168 indicated that they are utilized most rapidly at the time of maximum protein synthesis. In this thesis the author has demonstrated that RNA-polyphosphate complexes are utilized very rapidly by synchronized Chlorella at a time shortly preceding nuclear division. 0n theoretical grounds a complex of RNA, which is known to be involved in protein synthesis, and polyphosphate, which contains "high-energy" phosphate anhydride bonds, should be an ideal molecule for protein synthesis. Cyto- chemical evidence has indicated that the RNA-polyphosphate complex, in 31:2, has protein attached. This protein may have a structural function, but it could also have an enzymatic role. As a result of the metabolic utilization of the "undenatured" RNA-polyphosphate two types of material were produced. One seemed to be composed of RNA units with many short polyphosphate chains covalently attached to each RNA molecule. The other material was composed of an assortment of free polyphosphate molecules of various lengths. The polyphosphates which seemed to be attached to RNA at this time apparently have one end free and are probably esterified to ribose moieties at the other end. It is possible that both this RNA-attached polyphosphate and the free polyphosphate are next converted to ATP by an ADP- polyphosphate-phosphotransferase type of enzyme. SUMMARY Evidence was obtained to indicate that polyphosphates constitute about one-third of the total phosphorus in the actively growing Anabaena cell. The incorporation of radioactive phosphate into the polyphosphate of Anabaena was rapid. A material containing polyphosphate and RNA could be isolated by relatively mild techniques. This material was free from serious contamination by DNA, protein, amino acids, carbohydrates, or other phosphorus-containing com- pounds. ChromatOgraphy on DEAE-cellulose columns resulted in the separation of RNA-polyphosphate fractions which had different base ratios. Changes in the ratio of RNA to polyphosphate were found to occur as Anabaena cultures aged. These changes were interpreted on the basis of increased shading in older cultures and the accumulation of cells at one stage of the life cycle in dense cultures. In the case of synchronized Chlorella cultures a definite sequence of changes in the fractions of RNA- polyphosphate took place. During the first nine hours of light a build-up of RNA-polyphosphate complexes occurred and these complexes exhibited only trace amounts of meta- chromasy. Then between nine and 12 hours of light a sudden decrease in the amount of RNA and polyphosphate took place 169 170 accompanied by a change in the metachromatic characteristics and dialysis preperties of the remaining material. The different fractions of RNA-polyphosphate changed independently during the life cycle. When chloramphenicol was added at the crucial nine hour light stage, the decrease in RNA was less pronounced and several of the remaining RNA-polyphosphate fractions maintained their previous characteristics. In the dark phase of the synchronization cycle a build- up and decline of RNA took place without a corresponding change in polyphosphate. The ratio of RNA to polyphosphate of Anabaena in the middle part of the growth curve and of Chlorella in the light phase of the synchronization cycle was constant. Calculations based on this ratio and the micromoles of ribose per unit of RNA resulted in a figure of 15 polyphos- phate-phosphate units per RNA-nucleotide unit. This ratio was interpreted as being significant since it was essentially the same during synthesis and utilization of the complexes in both organisms. The larger complexes found in the 0 to 9 hour light period of the Chlorella synchronization cycle exhibited the following preperties: 1. large change in metachromasy upon dialysis against distilled water 2. large change in column chromatography preperties upon dialysis against distilled water 3. no release of metachromasy after freezing, boilin , dialysis against 0.2 M NaCl at pHs between 6 and , or incubation with EDTA. 171 h. hyperchromicity upon boiling 5. effects upon the metachromasy of the dialyzed complex by polyphosphatase, RNase, and snake venom.phosphodiesterase The data obtained were interpreted to indicate that these complexes involve a system of hydrogen-bonds or salt-bridges and also a system of covalent bonds. f '3 BIBLIOGRAPHY 1. 'Acs, G., Biro X. S., and Straub F. B., Hung. Acta Pfl2810 e g, 6“ (19u9)e (Ce An £2, 8616a e 2. Agroskin, L. S., Korolev, N. V., Kulaev, E. S., Mesel, M. N., and Pomoshnikova, N. A., Doklad , Acad. §g_i_., U.S.S.R. 131, 114110 (1960). 30 Albaum, He Ge, md 08111., Me, APCh. Biocheme 2:5,, 158 (19h7). h. Albaum, H. 0., Schatz, A., Hulner, s. H. and Hirshfeld, A., Arch. Biochem. 29, 210 (1950). 5. Anderson, I. C., and Robertson, D. 8., Plant Physio . 25, 531 (1960). "_— Belozersky, A. N., Con r. Intern. Biochem. Resum'es Commune?) 3° 00 r. BrusseIs _l, 955, 78. (0. I. 59 I562 g 1 e 0 1 0‘ e 70 Belozerski, Ae Ne, and Kulzev, Ie Se, 8100119310 (Russia 22, Elf-(1957). (English translation). 8. Benson, A. A., Bassham, J. A., Calvin, M., Goodale, T. 0., Haas, V. A., and Stepka, N., g. _A_m. Chem. Soc. lg, 1710 (1950). “'— 9. Berg, 0. 0., Anal: . Chem. 30, 213 (1958). 10. Blout E. R. and Anderson A. Biochem. et Biogfls. Act; g, 131 (19514). ' ' "—— "" 11. Bradley D. F. and Rich, A. J. Am. Chem. Soc. 78 5898 (195603 ' " "‘ """" ""' ' 12. Bradley, D. F., and Wolf, M. K., Proc. Natl. Acad. Sci. 0.3. 35, 9M. (1959). "“"' """ *— 13. Braun, A. D., and Nemchinskaia, V. I.., Biochem. (Russia) 22, 3140 (1958). (English translation). 114. Bringmann G. Zentralbl. Bakt. ARE. 3;. Orig. 1 6 1:93 (1931).' ‘5‘" 172 173 15. Brock, T. D., and Brock, M. L., Biochem. 23 Bioghys. Acta 33, 27h (1959). 16. Brown, A. R., and Webster, G. C., _Aln. _J_’. _B_0_t_. A2. 753 (1953). 17. Brown, D. M., and Todd, A. R., J. Chem. Soc. 20h0 (1953). """' "'" 18. Brown, F., and Cartwright, B., Biochem. et Biophys. Acta 3 s 3&3 (1959)e 19. Brown, G. L., and Martin, A. V., Nature 116, 971 (1955). ""‘-' 20. Bukovitch, E., and Belozersky, A. N., Doklad , Proc. Acad. Sci. U.S.S.R. 12Q, 11h? (I959). 21. Chargaff, E., and Davidson, J. N. (Editors) The Nucleic Acids, V01. 1, Academic Press, New York, 22. Charles G. Internatl. Geol. Congr. 12 163 (1953). (Biol’Abs: W).—‘ ’ 23. Chatterji, A. C., Bhargava, H. N., Tewari, K. X33, and Krishnan, P. 3., Arch. Biochem. Biophys. 179 (1951). 20. Chayen, R., Chayen, S., Roberts, E. R., Biochem. et Biophys. Acta 16, 117 (1955). 25. Colowick, S. P., and Kaplan, N. 0. (Editors), Mgthods in Enz molo , Vol. III., Academic Press, New York, 1955, p. 880. 26. Corbridge,‘D. E. C., and Lowe, E. J.,‘g. Chem. Sag. 1493 (1951;). ‘— 27. Cox, R. A., Jones, A. S., Marsh, G. E., and Peacocke, A. R., Biochem. 33 Bioghys. Acta g;. 576 (1956). 28. Damle, S. P., and Krishnan, P. 8., Arch. Biochem. 31021118. Q, 58 (195(4). 29. De Lamirande, G., Allard, C., and Cantero, A., ‘1. Biol. Chem. ggy, 519 (1955). 30. Doty, P., Boedtker, R., Fresco, J. R., Haselkorn, R. and Litt, M., Proc. Natl. Acad. Sci. Q5, h82 (1959). 17b 31. Dounce, A. L., Enzzmologia.1§, 251 (1952). 32. Dounce, A. L. and Ka , E. R. M., Proc. Soc. E'g. Biol. Esi- é , 321 {1953). (c. 1711,1357 8-1953). 33. Ebel, J. P., Thesis, Fac. Sci. Strasbourg (1951). (Biol. ug.—1.1621953). 3h. Ebel J. P., Bull. Soc. Chim. Biol. 3 , 321 (1952). (C,.A.y1,'5'51')'.—_ "‘“m ‘1‘ 35. Ebel, J. P. and Colas, J., Co . Bend. 2 , 173 (1951;). (c. A. gt}, 1350931792" """'" ‘2‘ 36. Ebel J. P. and Miller 8. Ex t1. Cell Research 1 21'(1958)'. ' ' _L """" "S" 37. Esposito, R. G., and Wilson, P. W}, Biochem. gt Bioghzs. Acta.§§, 186 (1956). 38. Fiske, C. H., and Subbarow, Y., J} Biol. Chem. 66, 375 (1925). """"" """"' 39. Flynn, R. M., Jones, M. E. and Lipmann, R., ,1. Biol. Chem. 211, 791 (1951.). ’40. F088, 6’. E0, BHCteriolo MO g2, 1’48 (1956). kl. Erick, G., Biochem. 23 Bioghzs. Acta 12, 352 (1956). h2. Fuller R. C., and Anderson, I. C. Plant thsiol. SuEEl.';g, xvi (1957). , h3. Gierer, A., and Schramm, G.,‘Ngture.111, 702 (1956). uh. Goksoyr, J. and Klungsoyr, L., Acta Chem. Scand. 13, 1497 (1959) . "“" ‘"" ’45. Gosselin, R. E., ROthBtein, A., Miller, G. J., and Berke H. L. J. Pharmacol. Exgtl. Therag. 106 180 (i952). ' " —"' h6. Gosselin, R. E., Tidball, C. S., Megirian, R., Maynard, E. A., Downs, W. L., and Hodge, H. C., J. Pharmacol. Exgtl. TheraE. 193, 117 (1953). (c. 1791, W. Ln. thte, H., g. Naturforschg. 8b, 173 (1953). h8. Griunberg-Manago, M. V., Biochem. (Russia) 23, 287 (1958). M9. 50. 51. 52. 53. 5h. 55. 56. S7. 58. 59. 60. 61. 62. 63. 6h. 65. 66. 175 Grunze, H., and Thilo, E., Naturw. 5, 26 (195k). Habergann, U., Biochem. 32 Bithzs. Acta 32, 297 19 9 . Harald, F. M., mderation PI‘OC. E, 956 (1959). Harvey, H. W}, Egg, Biol. Assoc. 22, 205 (1937). Heller, J., St. Karpiak, and Zubikowa, 1., Nature Herbst, F., Ber. Deutsch..§gg.‘éé, 283 (1953). (Biol. Ab57‘102287‘1955). Hoffmann-Ostenof, 0., Kenedy, J., Keck, R., Gabriel, 0., and Schonfellin er, H. N., Biochem. 33 Bithys. Acta 1 , 285 (l95h). Holley, R. N., and Doctor, B. P., Federation Proc. .229 3h8 (1960). Hurlbert, R. B., Schmitz, H., Anne, F., and Potter, V. R.,'g. Biol. £22m. 209, 23 (195k). Hutchinson, G. E.,'A Treatise on Limnologg, vol. g., In elman, B., and Malmgren, E., Acta Chem. Scand. Q, 8 M78 (1950). Ishihara, R.,.g. Biochem. (Japan) AZ, 196 (1960). Iwamura, T., and Myers, J., Arch. Biochgm. Bioghys. §H; 267 (1959). Iwamura, T., Biochem..g§ Bioghy . Acta Q2, 161 (1960). Juni, E., Kamen, M. D. Rainer, J. M., and Spiegelman, 8.. Arch. Biochem. l_, 387 (19h8). Katohman, B. J., and Van Wazer, J. R., Biochem. _e_t_ Bioghzs. Acta $3. hhS (l95h). Katchman, B. J., and Smith, H. E., Arch. Biochem. Bioghys. 15, 396 (1958). Rock, R., and Stich, H., Annals 2; Botan , N. .'gl, 612 (1957). 67. 68. 69. 70. 71. 72. 73. 7h. 75. 76. 77. 78. 79. 80. 81. 82. 83. 8h. 85. 176 Ketchum, B. H., and Redfield, A. C., J. Cell. Coma. Physiol. 3;, 281 (1919). King, E. J., Biochem..g.'§§, 292 (1932). Kifbyé K. 8., Biochem. g§,Bithys. Acta 31, 338 19 0 . Knaysi, G.,'g. Bact. 11, 532 (1959). Kornberg, A., Kornberg, S. R., and Simms, E., Biochem. g£_B102hys. Acta 29, 215 (1956). Kornberg, A., Advances ig_Enzzmologz 18, 191 (1957). Kornberg, S. R., Federation Proc. 1;, 2hh (195k). Kornberg, S. R.,.g. Biol. Chem. 218, 23 (1956). Kornberg, S. R., Biochem. 2; Bithz . Acta 26, 2914 ( 1957 ) . ""‘""" "'"" Kornberg, S. R., Lehman, I. R., Bessman, M. J., Simms, E. 8., and Kornberg, A., _._I. Biol. Chem. 233. 159 (1958). Kratz, W. A., and Myers, J., J. Amer. Botany H2, 282 (1955). "“ Kr s R. N., n. Egg, Plant Physiol. , 207 (11356). “AL 2 Krieg, A., Exgerientia 19, 20h (l95h). (Biol. Abs. 12932, 1955fi Kulaev, I. 8., and Belozersky, A. N., Biochem. (Russia) 22, 5&5 (1957). English translation). Kulaev, I. S., Belozersky A. N., and Mansurova, S. E., Biochem. (Russia) 23, 236 (1959). (English translation). Kulaev I. S. Kritsky M. S. and Kblozersky A. N. Biochem. (Russia) 35: 735 (1960). ‘ , ' Knnitz, M.,.g.'§gg. Physiol.‘25, h23 (1952). Langen, P., and Lies, E., Biochem. g. 339, h55 (1958). Lieberman, I.,.J. Biol. Chem. 212, 307 (1956). 87. 88. 89. 90. 91. 92. 93. 9h. 95. 96 . 97. 98. 99. 100. 101. 102. 103 . 10h . 105 . 177 Lindeberg, G. , and Malmgren, B., Acta Chem. Scand. g, 27 (1952). """’ "" """"" Lindegren, C. C., Exgtl. Cell. 333. _2_, 275 (1951). Lisa, E., and Langen, P., Naturw. H6, 151 (1960). Liss, E., and Langen, P., Biochem. _Z_. 132, 193 (1960). Logan, R., and Davidson, J. N., Biochem. 93 Bioghzs. Acta 2 , 196 (1957). Lohmann, K., and Langen, P., Biochem. _Z_. 328, l (1956). Lowry, O. H., and Lopez, J. A., _J. Biol. Chem. 162, uzl (19kb). Lowry, 0. Ho, Rosebruah, N. J., Farr, A. L., and Randall, R. V., _J. Biol Chem. 19:2, 265 (1951). Lund, J. W. G., _J. Ecol. 38, l (1950). Mallete, M. F., and Lamanna, 0., Arch. Biochem. Bioghzs. 53;, 217 (1951)). Malmgren, H., 5.9.15.9. M. §_9_a_{1_<_1_. 6, l and 16 (1952). Mann, T., Biochem. _J. _3_§_, 339 (1914(4). Mattenheimer, H., Biochem. _Z_. 322, 36 (1951). Mattenheimer H. _z. Physiol. Chem. :30} 107 115 and 121; (1956): ""‘" ' ' ’ McElroy, W. D., and Glass, B. (Editors), Chemical Basis 91: Heredity, John Hapkins Press, Baltimore, 9P- Mudd S., Yoshida, A., and Koike, M., _J_. Bact. 2 (1958). ""'"' 12' Muhammad, A., Rodgers, A. and Hughs, D. E., 3. Gen. Microbiol. 39, [(82 (1959). Niemierko, S., and Niemierko, W., Nature 166, 268 (1950). Nihei, T., ,1. Biochem. (Japan) 142, 2115 (1955). Nihei, T., _cI. Biochem. (Japan) M), 389 (1957). 106 . 107 . 108. 109. 110. 111. 112. 113. 11h. 115. 116 . 117. 118 . 119. 120. 121. 122. 123. 121). 178 Pierpoint, W. 5., Biochem. _J. 6_5_, 67 (1957). Bioghys. g}, 238 (1958). £25. 2&75 (1960). Ho Go, is 8101. Chain. Rafter, G. W., Arch. Biochem. Rafter, G. W., J. Biol. Chem. Razzell, W. E., and Khoranna, agg, 2105 (1959). Reisner, G. S., Goring Plant Physiol. 35, [48 R1011 A. and DaV13 D. R. J. M. Chem. 80°. 8 35138 (1956). ’ ’ "' "" "" "— 1" Robinson, W. W. Jr., U.S. 2657178 and 2657179, Oct. 27, 1953. (c. A. 8, 918e). Rothenbach E. F., and Hinkelman, St., Naturw. A}, 555 (195$). —— Sager, R., and Salokar, M. , Nature 162., 98 (1958). R. K. and Thompson, J. F., (1960). Sall, T., Mudd, S., and Davis, J. C., Arch Biochem. Biophys. _6_9, 130 (1956). Sall, T., MUdd, 8., and Takagi, A.,.g. Bact. Z6, oho (1959). Sansoni B., and Kement, R.,.Angew. Chem. 66, 598 (1958). Sarkar, N. K., Dounce, A. L. and Lacognata, E., Federation Proc. 18, 1251 (1959). Schmidt, G., Hecht, L., and Thannhauser, S. J., _J. Biol. Chem. 178, 733 (19149). Schmidt, G., Seraidarian, K., Greenbaum, L. M., Hickey, M. D., and Thannhauser S. J., Biochem. 33 Bioghys. Acta g9, 135 (1956). """" Schmidt, R. R., Federation Proc. 19, 126 (1960). Schweiger, H. G., 12. 395 (1960). Shuzo, A., Yokoi, H., and Goto, H., _g. Biochem. (Japan) A5. 959 (1958). ""“" Smith, J. D., and Markham, R., Biochem. J. (1950). and Bremer, H. J ., E. Naturforschg. 6, 509 125. 126. 127 . 128 . 129 . 130 . 131. 132. 133. 13A. 135. 136. 137 . 138. 139 . 1&0 . 1141. 112. 1&3. 1111;. 179 Sommer, A. L., and Booth, T. E., Plant Physiol. 13 199 (1938). ’ Soi'okénS C., and Myers, J., _J_'. 223. Physiol. 69, 579 19 7 . Stange, L., Bennett, E. L. and Calvin, M., Biochem. _e_t_ Bithys. Acta 31, 92 (1960). Starr, R. C., Llozdia _12, 129 (1956). Stich, H., g. Naturforschg.'§2, 36 (1953). StiCh, Ho, _2_0 Naturforacgfio 10b, 28]. (1955). Szulmajster, J., and Gardiner R. 0., Biochem. 9.2 Bithys. Acta 39, 165 (1960). Takeda, Y., Hayashi, S., Hakagawa, H., and Suzuki, F., _J_'. Biochem. (Japan) 36, 169 (1960). Tamiya, H., Iwamura, T., Shibata, K., Hase, E., and Nihei, T., Biochem. g_t. Biophys. Acta 3:3! 231 (1953). Taussig, A. and Creaser Bioghy . 8 . h36 (1959). Tewari, K. K., and Krishman, P. 8., Arch. Biochem. Biophys. 82, 99 (1959). E. H., Arch. Biochem. Thilo, E., Grunze, H., Hammerling J., and Werz, G., ‘g. Naturforschg. 11b, 266 (195 ). Thilo, E., Natmo ‘99" 367 (1959). Thomson, J. F., and Sato T. R., Arch. Biochem. Bioghys. 82, 139 (1960). Vizoso A. and Burness A. T. H. Biochem. Bio h s. Res.,Comm. g, 102 (1960). ' """"— _J‘L Wassink, E. C., and Romback, J., Proc. Koninkl, N_e_d_. Akad. Wetenschgp. 7c, 1493 (19511). Webb, J. M., g. 8131. _g_h_e_:_r_n. _2_39, 1023 (1958). Wiame, J. M., _J. 193‘ £1313. 692. 69, 31116 (191(7). Wiame, J. M., _J. _B_1_9_];. £1311. m, 919 (191-(9). Widra, A., _J. @3333. Z_8_, 66h (1959). 1&5. 1A6. 1&7. 1h8. 1&9. 150. I151. 152. 153. 15h. 155. 156. 157. 180 ‘Williams, E. J. and Fbster, J. F., J. Am. Chem. Soc. 82, 2A2 (1960). M., Nature 17 WinderL)F., and Denneny, J. , 353 (195 Winder, F., and Denneny, J. M., Nature 17 , 636 (1955). ""“' Winder, F. _15, i (1956 Winder, F. G., and Denneny, 17. 573 (1957). Wintermans, J. F. G. Wetenshgp. 57c, 57h W( Wintermans, J. F. G. M., Mededelingen van De Landbouwhogeschool Te WEgeningenZNe de rlafia 55. 69 (1955). YOShidB, A., 3310 P8. era .C01lo' Geno Educ. 1111170, Tokyo_111,151’( Yeshida, A., and Yama taka, A., J. Biochem. (Japan) Yoshida, A.,-g. Biochem. (Japan) 62, 163 (1955). Yoshida, A.,IJ. Biochem. (Japan) 62, 381 (1955). .3 and Denneny, J. M.,.J. Gen. Microbiol. J. “-9.29 Gen. Microbiol. Proc. Koninkl. Ned. Akad. Zaetseva, G. N., Belozersky, A. N., and Frolova, L. V., Doklad Acad. Sci. U.S. SR .1 2, 950 (1960). Zaetseva, G. N., and Belozersw Acad. Sci. U. S. S .R. 132,950 970 o No, Daklad , g(1960). 10015; US": Pl“ "x sf"! .7 a K‘ 'g a. q :3