Ill! ll 118 370 THS :‘ '. l:- m; 1“; 5” . .ani;¢3-s- 1;".3 » - "a," ’€ "4.." . ~ . y " I... 3- 'I.‘ did“ -.\\::;;H ‘1. v ‘ ‘ 9 was“: .as :43 i¢\"03 ,y:, ‘.' _ . , . ,;,y--z.1 e.- '11 H 3; .7 :" ‘- ' «5'.- ~73.“ ;-\:.- jiV’x :-----J~"‘"¢ "’ ,2“; ' '1 A : ‘6‘ ’\ 'Y' rt. :3 ., \- ,_ iv ’o “a: a _ ..t. dfl‘ Vt . . ‘. ‘. ‘3‘ ‘K. £g ¢ J‘ C’ --"'!!:.’i!l!’)‘"n.': '1)! 2321 “thong-a... .- \ . ‘. ,‘5 4-, .3 ~14 ".- V. :‘ '» ‘fi . . ,1 g 9; , . J. .r 9"} ‘7' k g . .. $- ' ._V . u: z o ' ’ ; ‘. ‘I f ‘_ ; ‘- ‘ ' ‘ -_ , .2; g" .\ - '; .-: kt? -‘- :- ' i ‘ Q U -" ‘ ' ' . ,t— ' ‘ a . - iv. _ “t! , L a . o o ‘ V " .'1 '7‘ -‘\’. >J‘ .1 u. (3.1 LIBRARY Michigan Sm» University MtCHIGAN 31m UNIVllgm R E C B I V E D DI". 0' CM‘YI‘ #7 was STUDIES OF PYRD’HDINE BIOSYNTIESIS BY NEUROSP-ORQ CR’LSTSA 1298 BY Olga. V1 ta]. Mi 1181‘ A 175315 Submitted to the College of Science and Arts of Michigan State UniVersity of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemistry 1959 1 Olga Vital Miller ABSTRACT To study the pathway postulated (6) for pyrimidine biosynthesis by the.mutant.Eeurospora crassa 129§,dihydrouridine and ureidopropionic riboside were prepared and their effect upon the growth of the mold ob- served. Neither of these two compounds supported the growth of the mold when they were used as sole supplements to the basal nutrient medium. Ureidopropionic acid riboside had no effect upon the growth of the mold on uridine or on aminobutyric acid, but dihydrouridine caused greater than additive effect upon the growth of the mold on these com~ pounds. These experiments do not provide evidence in support of the path- '; however, the possibility exists, that the mold.was not able to phOSphorylate dihydrouridine and ureidoprOpionic riboside, therefore the results cannot be taken as conclusive evidence against the pathway. Several compounds which were found to be non-supporters of growth of the mold in the normal basal medium (pH of 5.6) were reinvestigated again in basal media having the pH of h and 3. Of these compounds succinic acid and glycine supported growth at the pH of h and alpha- ketobutyric acid supported growth at the pH of 3. Lowering of the pH had no effect upon the growth supporting ability of acetic acid, pyruvic acid, beta—alanine, DL-aSpartic acid, gamma—aminobutyric acid, DL—alpha-amino isobutyric acid and ornithine. The finding that alpha-ketobutyric acid supports the growth of the mold leads to the suggestion that alpha—aminobutyric acid might be able 2 019a Vital Killer to convert to prOpionic acid through the intermediate alpha-ketobutyric acid, without necessarily passing through homoserine and.beta—hydroxy propionic acids as intermediates. ACKN GREENE»? T The author wishes to express her appreciation to Dr. James L. Fairley for his interest, encouragement and guidance in completing this project. She also wishes to express her gratitude to the Atomic Energy Commission for a grant supporting this work. TABLE OF C NTENTS Page Ev; TmDUCHGJ I I I I o I o I I I I I I o I I o o I I I I o o 1 EXPERU 12:: T8 m: D IESWLJI‘S I I I I I Q I I I I I I I I I I I o 7 Effects of dihydrouridine and ureidopropionic acid riboside on the growth of y, crassa 1298 . . . . . 7 Preparation of dihydrouridine . . . . . . . . . . . 7 Demonstration of purity of dihydrouridine . . . . . 8 Preparation of ureidopropionic acid riboside . . . . 11 Demonstration of the purity of the ureidopropionic aCld r1b031dz O I O O I O O I O O O O I O O O O 0 ll Organism . . . . . . . . . . . . . . . . . . . . . . 12 General growth procedure . . . . . . . . . ... . . . 13 Results of testing for growth . . . . . . . . . . . lb Effect of pH on growth . . . . . . . . . . . . . . . . . l7 nnmmfinm ... ... ... ... ... ... ... ... . 21 some! 2h Bmummm O C I C I O O O Q 0 O O O 0 O O I O O O 0 O O O 25 .... LIST OF TABLES TABLE Page I The composition of the basal nutrient medium . . . . 13 II Results of the growth.procedure for dihydrouracil and ureidoprqpionic acid riboside . . . . . . . . 15 III Effect of dihydrouridine and ureidopropionic ribo- side upon the growth of the mold on uridine . . . 16 IV Growth of the mold.§, crassa 1298 on dihydrouridine and.ureidopropionic riboside when the inncculation is by mycelial fragments . . . . . . . . . . . . 13 V Relative rate of growth of the mold at different pH values with various supplements . . . . . . . 20 IRTRODUCTIQN The biosynthesis of the nucleic acid pyrimidines has been studied by many investigators for a number If years, and while much experimental evidence has been collected, there remain mamr unanswered questions. One preposed route of pyrimidine biosynthesis, the Liebermann-iiornborg (l) scheme, is accepted by many as a pathway both for pyrimidine form- ation and degradation. The sequence of compounds in this scheme is the followings ' maloacetic Midi—3:19 aspartic acid' ’ ureidosuccinic acid H dihydroorotic acid uridine—Shphosphateiw-torotidine~5'-phos.phate v=orotic acid H uridine-S'-triphosphate e— ; cytidine—5'~triphosphate \ nucleic acid pyrimidines This is just one possible pathway; under different conditions, and in other organisms other pathways might assume importance. Pink and her coworkers (2) demonstrated an enzyme system in rat liver which degrades thymine to beta~sminoisobutyric acid. The conversion of beta-ureidoiso- butyric acid to beta-aminoisobutyric acid appeared to be irreversible, 2 but dihydrothymine and beta-ureidoisobutyric acid were interconvertible. Canellaisis (3) carried out similar experiments with uracil, di- hydrouracil and ureido—propionic acid. The results were similar to those of the previous investigators, except that the overall conversion of uracil to bets~alanine was not demonstrated. The rate determining step, the slow step both with uracil and thymine, was the first step— the conversion of the pyrimidine to dihydrouracil or to dihydrothymine, while the rate of degradation of dihydrouracil and ureido-propionic acid was found to be much higher. It seems possible that these reac- tions can be reversed under appropriate conditions to lead to the syn~ thesis of pyrimidine compounds. Fairley (h) demonstrated the growth of pyrimidineless En.2£§§§2 mutants on threonine and alpha~aminobutyrate, and showed the more than additive effect of the presence of alpha-aminobutyrate on the growth of the mold.with uracil and uridine. Threonine had the same stimulatory effect, while aspartic acid had no effect. There was no stimulatory ef- fect by any of these compounds on the wild—type strain of N. crassa. This phenomenon appears to be associated with yet another mechanism for pyrimidine formation, a mechanism distinctly different from the Lieber- mann-Kornberg preposal. Herrmann and Fairley (5) used aminobutyrate-B-C (1h) in the basal growth medium and found that the labeling of the pyrimidines produced by the mold was much greater than any of the other constituents. The results were not completely conclusive as far as the utilization of the number 3 carbon, as the compounds isolated were only 1/10 and 1/15 as active as the administered aminobutyric acid. However, the amino— butyric acid reisolated from the mycelium also showed great dilution of the isotopic carbon, indicating that while the mold did require aminobutyric acid for growth, during the growth it.produced.more amino- butyric acid or a closely related derivative. Boyd tested a great variety of compounds in search for ones which are utilized f0r'pyrimidine biosynthesis. She divided these compounds into four groups: the uracil group which contains compounds which were known to be intermediates or could be intermediates in the uracil and thymine degradation; the aspartic acid group, which are intermediates or could be intermediates in the Liebermann-Kornberg scheme; the alpha- aminobutyric acid group; and the prepionic acid group. In the uracil group, uracil, dihydrouracil and ureidopropionic acid supported growth. There was a time lag in the growth curve'of dihydro- 'uracil and an even greater leg for ureidopropionate, leading to the conclusion that these substances are not direct intermediates in the synthesis of nucleic acid.pyrimidines, but that they can be converted to such intermediate 3 . The compounds in the aspartic acid group did not support the growth of the mold, which again confirmed.previous experimental results that the mold uses a different route from the one outlined in the Liebermann—Kornberg scheme. Only alpha-aminobutyric acid, homoserine and threonine were used by the mold from the aminobutyric acid family. .Alpha~ketobutyric acid and alpha-hydroxybutyric acid did not support growth. Propionic acid, betawhydroxypropionic acid and methylmalonic acid did support growth while other acids in the propionic acid family, succinic acid, pyruvic acid, and acetic acid.proved to be inactive. Time course studies by Boyd (6) showed that the biosynthesis from propionic acid and alpha-aminobutyric acid follow different paths from ..I‘EszEJo-‘Il’il [urhl I H .... A . INDIE. a yr . 4.? V .' b that used by the uracil group. The similarity of the growth pattern with aminobutyric acid and propionic acid and also their similar in- hibition by arginine as shown by Fairley (h), led to the suggestion by Boyd (6) that they are utilized by the same route, with a common inter- mediate. Aminobutyric acid, homoserine and threonine all support growth in similar manner, as was shown by Fairley (L). Homoserine and threonine are possibly inberconvertible through a vinylglycine inter- mediate (7) which would also give alpha-aminobutyric acid upon hydro- genation (3). The pathway proposed by Boyd (6) for pyrimidine synthesis in the mutant, Neurospora cmeea 1298 is: prep 1 onate 5:22: prop-l any 14CoA §======3 be ta—a lany l~CoA l V be ta—alanyl-CoA-ribotide U dil'zydrouracil ribotide '--—-—-~‘- beta-ureidopmpionyLCoA ribotide uracil ribotide * , fl; nucleic acid pyrimidines. Boyd's scheme also proposed the following introduction of alpha-amino- butyric acid into this scheme: alpha-eminobutyric acid homoserine =12: alpha—l-zetmgannna—lrydroxybutyric acid beta-hydroxypropionic acid .4\ beta-alaxwl—Cdl. 5 The reason for this suggested mechanism, rather than the simpler pro- duction of propionyl-COA from aminobutyrate by deamination and de- I carbonylation, was the experimental evidence that alpha-ketobutyric acid did not support the growth of the mold, while the fact that hy- droxypropionic acid supported growth of the mold made the hypothesis a more likely one. Mokrasch and.Grisolia (9) have reported.that in rat liver prep~ arations, uridylic acid, dihydrouracil ribotide and ureidopropionic acid ribotide are utilized for nucleic acid pyrimidine formation, but uridine and dihydrouridine are not used as readily. The phOSphorylat- ing mechanism.therefore appears to be absent in the rat liver homo- genate. The experiments described in this thesis were initiated to provide further evidence concerning the route of pyrimidine formation in the mutant organism,‘Neurozpona crassa 1293, with emphasis on two major aSpects. It seemed possible to test the hypothesis of Boyd for the biosynthetic route by preparing several of the suggested intermediate compounds and testing them for growthnsupporting abilities. Compounds with readily ionizable groups, such as the phosphate groups of the ribotide intermediates, will not pass through the mycelial membrane. These compounds could not be tested directly. However, in comparison with uridine, it might be expected that the mold could absorb the ribosides and.phosphorylate them inside the mycelium. Accordingly it was decided to prepare two of the ribosidic compounds, dihydrouridine and.ureidopropionic acid riboside, corresponding to two of the ribo~ tides of Boyd's scheme and test them for activity with the mold. 6 Boyd's conclusions concerning some of the early reactions of her proposed mechanism were based solely on the results of growth tests at pH 5.6. It seemed possible that permeability considerations, partic- ularly with moment to some of the more acidic compounds tested, could have led to erroneous conclusions in that some of the compounds may not have been able to pass through the mycelial membrane barrier in the ionic f0.-. . “ibis possibility has been tested in the present work. TL! h; *;uI‘-' EXPERIIMES AN D IESULTS Effects of dimidrouridifine ancijxrewidopropionic acid riboside on the growth of E. crasse 1298. Preparation of Dil'vdrouridine Dihydrouridlne was synthesized from uridine by a slight modifica- tion of the procedure of Green and Cohen (12). An all glass aemimicro hydrogenation apparatus was used. The burette was filled with hydrOQen from a reservoir after repeated flushing of the apparatus with hydro- gen. The preeeure of the system was atmospheric. The boat containing 90 mg. of a rhodium on alumina catalyst with 250 mg. of uridine in to ml. of water was simi-zen by an electric shaking device during the hydrogenation. The catalyst was first saturated with hydrogen, the uridine was then added and the hydrogenation was continued until the hydrogen uptake ceased, a period of about 50 minutes. The hydIOQen uptake corresponded to the timoretical 100 percent hydrogenation. Levane (13) reported the production of dihydrouridine as an oil, but Cohen (12) obtained it ea an amorphous compomd after repeated evaporation of the hydrogenated compound. from absolute alcohol and precipitation of the solid from the alcohol solution by absolute ether. Most of the growth studies of the present report were made by using dihydrouridine as an oil, assuming approximately 100 percent yield from uridine. Dihydrouridine, however, was finally obtained as a white crystalline solid after several attempts. Some of the pre- vious growth experiments werethen repeated and gave results identical to those obtained by the use of the oil. A.‘ o..- H\.ru...|... nu. a 1‘.. Ir ... . , .w Ix a l. i -L in 8 Demonstration of Purity of Dihydrouridine That the oil obtained in most experiments was pure dihydrouridine was deno:streted.by shoeing that no uridine remained, that no free sugar was present, that no free ureido group was present, that a group was present which.was converted to the ureido group upon treat- ment with alkali and that no free dihydrouracil was present. The same methods were used to show the purity of the crystalline dihydrouridine. To show the absence of uridine from the hydrOgenated.produdb a Becknann Spectrophotometer was used. Atra wave length of 260 nu uridine absorbs radiation. The molar extinction coefficient is 10,000. Fif— teen mg. of dihydrouridine dissolved in 3 m1. of water gave an absorb- tion of O.h02. herefore the maximum amount of uridine was less than 0.01 ng/nl., or less than 0.2 percent of the starting amount. Both the oil and the crystalline pr-,aration of the dihydrouridine gave identical results concerning the presence of the maximum amount of uridine. To show the absence of free ribose in the preparation aniline hydrOgen phthalate color reagent (lb) was used. The reagent was pre- pared by dissolving 0.9 g. aniline and 1.65 g. phthalic acid in 100 ml. of n~butyl alcohol soturetcd.with water. Fifty micrograms of the dihydrouridine preparation was placed on Whatman No. 1 paper with micropipet e, allowed to dry, and then sprayed with the aniline hydro~ gen phthalste reagent. The paper was then placed in a ventilated oven at 165°C. for five minutes. No color developed. The same pro- cedure was repeated with different portions of ribose solutions and .1- n ., . ”an: even one microgran ribose produced.pink color with aniline hydrogen phthalate. Therefore, the ribose was still in the glycosidic form. The absence of a ureido group, but the presence of a group which can be easily converted to a ureido group was shown by the use of the characteristic color reaction of the ureido group with.pmdimethyl - aminobeneal‘ehyde (15). The color reagent was prepared.by'dissolving- l g. of pudimethylaminobenzaldehyde in a solution of 100 ml. of ethanol and 10 ml. of concentrated hydrochloric acid. Portion: of the dihydro~ uridine preparation corresponding to 30, 70, and 150 micrograms of di~ hydrouridine were applied with a micropipette attached to a hypodermic syringe to two stripe of Whatman No. 1 paper 3.5 inches from the end and one dimensional paper chromatography was carried out. The paper was stretched between books and dried with a hair dryer between the addition of portions of the solutions to help maintain the size of the spots to a small circle. After complete application of the solutions, the papers were folded 1 inch from the origin, and then again 2 inches from the origin in the Opposite direction. The one inch flaps were placed in a glass trough and the papers were passed up and over 91885 rods in such a any that the second fold coincided with the glass rod and the paper hung straight down from this point. The solvent used nae a mixture of t—butyl alcohol, seawbutyl alcohol and water in the ratio of 12535.6. The glass troughs were placed in a 12 X.2h inch battery jar'which contained 100 ml. of solvent in the bottom, and then the jar was covered.with a glass plate. After an hour's waiting per— iod 75 ml. of solvent.uns added to the glass troughs through the holes of the glass plate and the system remained covered until the solvent 10 almost reached the end of the paper strips. The papers were then hung to dry in the hood, the drying being facilitated by the use of an elec— tric fan. One of the strips of paper was sprayed with 0.5 N sodhzm hydroxide and allowed to stand for an hour to dry. The other paper was not treated with sodium hydroxide. Both strips then were sprayed with the p—dimetrylaminobenmldehyde solution. On the paper which was treated with sodium hydroxide yellow spots developed upon stand- ing at an R1. of 0.50, which indicates the presence of a ureido group produced by basic hydrolysis, but no colored spot deve10ped on the other paper, which showed the absence of the free ureido group from the original dihydrouridine preparation. The Rf value is defined as the ratio of the distance of migration of the compound being chromato- graphed to the distance traveled by the solvent. To show the absence of dihydrouracil from the preparation, 50 micrOgrams of dihydrouracil and 100 micrograms of the prepared dihydro- uridine were spotted on a strip of paper and one dimensional paper chromtogmpby was carried out as in the procedure above. The dried paper was sprayed with 0.5 N sodium hydroxide, dried again and then sprayed with the p~dimethylaminobenzaldehyde color reagent. The Rf values of dihydrouracil and dimrdrouridine were 0.36 and 0.50 respec- tively. The dihydrouridine preparation did not produce a yellow color Spot corresponding to the 0.36 value of dihydrouracil, which confirms that in the hydrogenation and isolation process the glycosidic linkage remained intact. 11 Preparation of Ureidopropionic Acid.Edboside Ureidopropionic acid riboside was synthesised from a portion of the dihydrouridine prepared hy the procedure described above. No at- tempt was mde to prepare ureidopropionic riboside in crystalline form, as the glycosidic linkage is quite unstable. A procedure has been re- ported by Batt (16) to obtain the barium salt of ureidopropionic acid, ' but at the pH of 2 as the synthesis is carried out, the ribose group of the ureidopropionyl riboside would not remain attached. Seventyfive mg. dihydrouridine in 25 ml. of water was allowed to stand a few hours with 25 ml. 0.1 N sodium hydroxide. The solution was neutralized‘to a pH of 7 with 0.1 N hydrochloric acid, the course of neutralization followed by a pH meter. The amount of hydrochloric acid used was 19.2 ml. and the total volume of the solution increased to 75 ml. {The approximate concentration of ureidopropionic acid ribo- side in the solution was 1 mg/nl. Demonstration of The Purity Of The Ureidopropionic Acid Riboslde lhe purity of the ureidoprOpionic acid riboside preparation was demonstrated by showing that no dihydrouridine was left, that no free ribose was present, and that the free ureido group was present. To show the absence of dihydrouridine paper chromatography was used again as described.previously with the same solvent. On one strip of paper dihydrouridine was spotted and on two other*papers the ureidoprOpionic acid riboside preparation. The papers remained l2 overnight in the troughs with the solvent. The lengths to which the solvent traveled was marked. After the papers dried in the hood, the paper on which the dihydrouridine was Spotted and one of the papers on which the ureidopropionic acid riboside was spotted were sprayed with the 0.5 1-4 sodium hydroxide solution and allowed to stand to dry for an hour. Then all three strips of papers were sprayed with the p-dimethyl- aminobenealdehyde reagent. Both strips of paper on which the ureido- prepionic riboside preparation was spotted developed only a single yellow spot upon the treatment with the p-dimethylaminobenmldehyde. The Rf values were identical, 0.30. The Rf value of dihydrouridine was 0.50 as was determined from the third paper. The results indicate the complete conversion of dihydrouridine to ureidOpropionic riboside. The absence of free ribose was again shown by the use of the hydrogen phthala’oe reagent. Organism Neurospora grassa 1298 was produced from a wild strain by Beadle and Tatum (10) using X-«rny treatment. Lorlng and Pierce (11) showed that the mutant will not grow on a simple basal medium of salts, sugar, and biotin, but it will grow upon addition of pyrimidine compounds. The mold was maintained on culture slants which were kept in a desiccator over a saturated solution of calcium nitrate to maintain the proper humidity for optimumlgrowth. The culture slants were pre- pared by dissolving 2 g. of agar and 100 mg. of uracil in 100 ml. of basal medium by the use of heat. Ten m1. fractions of the solution were transferred to Pyrex culture tubes. The times were stOppered with n La“. 3 5. rt If 13 cotton plugs and sterilized by autoclaving. The tubes were then placed on a slant and allowed to gel in that position to provide a greater surface for the mold to grow on. TABLE I TI‘IF. COMPOSITIW OF THE RASAL NUTRIENT MEDIUM Calcium chloride 1 9. Trace element solution 50 )11. Asmonium tartrate 50 g. Sodium tetraborate 8.8 g. Armonium nitrate 10 g. Ammonium molybdate 6.1.; 9. Potassium dihydrogen Ferric chloride 50.0 9. phosphate 10 g. Zinc sulfate Magnesium sulfate heptsbydmte 200.0 g. heptahydrate S g. Cupric sulfate 27.0 g. Sodium chloride 1 g. Manganous chloride h.5 g. Sucrose 101 g. Distilled water to 500 ml. Biotin 26 pg. Distilled water to 10 1. The mold was also maintained on culture slants in which 100 mg. alpha-aminobutyric acid replaced the 100 mg. uracil. The mold was transferred from tube to tube at two week inter- vals using standard sterile technique. ' General Growth Procedure The mold was grown in 125 ml. Erlenmeyer flasks to which 25 ml. of basal nutrient was added. The flasks were stoppered with cotton plugs, then autoclaved for a 20 minute period. When the compound which was tested for growth was heat stable, it was added before the autoclaving procedure to the basal medium and the complete solution was autoclaved. The labile compounds, dihydrouridine and ureidoprop- ionic acid riboside, were not autoclaved, but the solutions of these compounds were filtered through a sterilized, sintered glass, bacter- iological filter and pipetted with sterile pipettes into the autoclaved .P .1 ‘I z“ ‘ I-‘tm~‘ .. ,I I | . {147'}. ,1» ' 49: '5 in basal nutrient medium. The solutiOns in the cooled flasks were inoc- culated with spores of E, 233323 1293 suspended in sterile water. All solutions were run in triplicate, with a solution without any supple- ment to the basal medium running alongside of them as a blank. After h days in the incubator at 26° C, any mycelial pads formed by growth were washed.with distilled water, dried overnight in an oven at 50° C, and then weighed on a torsion balance. Results of Testing for Growth The results of experiments in which dihydrouridine and ureido— propionic riboside were sole supplements to the basal medium are given in Table II. The result of the growth of the mold on uridine is also given for comparison. The possible effect of dihydrouridine and of ureidopropionic ribo- side upon the growth of the mold in the presence of uridine was also ' determined. The results of these eXperiments are summarized in Table III. The enzymes of N. crassa 1293 involved in the formation of pyrim— idines by this new route are probably adaptive in nature. Boyd (6) showed this by demonstrating the time lag in the growth of the mold both on aminobutyric acid and on propionic acid. To see what effect the adaptive nature of the enzyme had upon the growth of the mold on dihydrouridine and on ureidopropiOnic acid riboside, a set of experi- ments was carried out in which the inocculation was done, rather than by a spore su3pension, by the use of a suspension of fragments of L- TX." ...: II . ‘u-E -,4:" 1 r; ‘P‘f‘ :V L -~~' -.-\1"_~.w(\- 7" awn-..- a. .:.o. &-*v "V . "V *3 w“- \—‘ h‘w 1 A L : --'s'-I:\.'B-rLr‘/' . k 4 ‘ ‘9’ I 1"...— U». :‘I" '(Fr’fi ..r:,- .I. 1"... i. .v tn f‘. mgr -l~vu-’--~J'-.J \J'V ..' T I ‘- t-O t '51 i .- -... -. .3- 313px?“ 4.1.... 220 Si“""‘.L"?3!‘. ‘— '\ ‘ :-v u; lune - or 9 .. ... a..'«. Qiagf jroe-rl\ll..g dl:"1f dz‘ouriiz.e .8 P‘. .. , f“ ‘1‘ £OF$C3J:.:4.':\1:.§$~JH of Supp le .. a A Vol. \‘1 C) F'.‘Jl*"“) ... )‘o Kr-“ )9 “H"! a s; (3 fl” ficg.lnulwfipt. I._,s.||. .....Ll‘ t)“. In. . i EFFECT OF DIHYDRQURIDIHE AND UREIDOPRDPICHIC RIBSSIDE UPON TH GROL'JTI‘I 05‘ T3115 MOLD ON URI 1331’. TABLE III U1 16 0.2 m3. uridine Simpletnents 0.1 m3. uridine Exp. 1 Exp. 2 No supplement 5.9 mg. 17.3 mg. 13.0 mg. Dil‘n'drouridine 0.2 mg. 703 my. lhos mg. 1306 mg. Dinydrouridine 0.5 mg. 21.1 mg. 16.5 mg. 31.1 mg.‘ Ureidoprooionic riboside, 0.2 mg. 5.8 mg. 12.5 mg. 11.1 mg. Ureidopropionic riboside, 5.0 mg. 5.6 mg. 11.6 mg. 11.2 mg. ”4‘ Q 3K1}. immfifimm'm "4—." v ." 5|: fiu-‘ ‘ I f ‘ 41..I.-.1«.£u7.: 3:819... ‘73. I. 1 ‘. fl». 1 . ‘ a, 1? mycelium which hadipreviously been grown on aminobutyrate. he results of these experiments are given in Table IV. The mycelium was grown in flasks containing 25 ml. of basal med- ium with 10 mg. of aminobutyric acid as the supplement. After five days of growth the myselium.was homogenized with an autoclaved Haring Blendor and 0.5 ml. of the homogenate was used to inocculate the so- lutions. Since the homogenate contains some aninobutyric acid, greater variation was expected in the growth of the myceliun, and therefore the eXperinent was carries out in quadruplicate. Effect ofgg! on Growth The optimum pH for the growth of Neurospora crassa 1298 is the pH of the basal medium as given in Table I, a pH of 5.6. Nucleotides and other strongly acidic compounds are not able to permeate the mem— brane of the mold at this pH. Some of the compounds which were found to be non-supporters of the growth of the mold at the normal pH were tested again at a pH of h and a pH of 3 to test the possibility that this negative result was due to the impermeability of the membrane to the compound. The inocculation and the procedure for growth was the same as previously described under the general procedure. At highly acidic conditions the growth of the mold is poor, and the mycelium difficult to collect and weigh. The amount of growth with different supplements was only estimated relative to each other by visual comparisons of the amounts of mycelium produced. -: ' ".- u ' 3.:‘4' Lina—WK) ”Ii-W '3'1 .; ._ ..-, y. .fm-‘ 18 TABLE IV In“; ens-rm or me new 3 . CRASSA 1298 or: mmmumnme AND '1 URBIDOPROPIOI‘JIC RIBOSIDE W} ‘23 THE INOCSULATION IS BY MYCEHAL FMGI‘MIS Concentration Average weight a J Supplement of of Supplement Hycelium no supplement 3.9 mg. aminobutyric acid 1.5 mg. 12.7 mg. dihydrouridine 3.0 mg. 19.1; mg. ureidopropionic riboside 3.0 mg. 3.1; mg. 19 The compounds tested were acetic acid, pyruvic acid, glycine, aSpartic acid, beta~alanine, gamma-aminobutyric acid, alpha~aninoi30~ flutyric acid, beta~aminoisobutyric acid, ornithine, alpha—ketobutyric acid, succinic acid, dinydrouracil, propionic acid and for comparison uracil. The compounds which did not support growth of the mold under any of these conditions were acetic acid, aspartic acid, beta~alanine, gamma-aminobutyric acid, alpha-amino~isobutyric acid, beta—aminoiso- butyric acid and ornithine. The remaining compounds are arranged in Table V according to their decreasing ability to support the growth of E, crassa 1298. 3%}...2 - H. .L RELATIVE TABLE V RTE OF.“ GROZJI'II OF THE HOLD AT DIFFEEEJT pH VALUES WITH VARIOUS SUPPLEMENTS 20 fiH 5.6 pH h PH 3 uraci l prepionic adid dihydrouracil glycine £1 succinic acidcz' alpha~ketobutyric acid (n growth) uracil propionic acid 9'— Succinic acid glycine dihydrounacilfia pyruvic acid alpha-ketobucyric acid (no growth) uracil c; alpha~ketobutyric acid propionic acid dihydrouracil glycine c: succinic acid (no growth) DISCUSSION The mutant, E, E£§§§E 1293, synthesizes the pyrimidines of the nucleic acids by a route other than the Liebermann-Kornberg scheme. Boyd did suggest that this route involves the conversion of propionate through beta-alanyl-COA, beta-ureidopropionyl—CoA ribotide, and through dihydrouracil ribotide to uridineMS’—phosphate. The present work in— volves the testing of this scheme by synthesizing compounds related to intermediates of this proposal, and observing their effect upon the growth of the mold. This result does not support the existence of this pathway. While these experimental results do not provide supporting evi- dence for the proposal of Boyd, these negative results cannot be taken as conclusive evidence against the proposed.pathway. Although the mold is able to phosphorylate uridine it may not possess the specific en- zymes needed to catalyze the phosphorylation of dihydrouridine and of ureidopropionic riboside. Hohrasch and.Grisolia (9) demonstrated the absence of such phOSphorylating mechanisms in rat liver homogenate. The phosphorylated conpounds, as has been noted, cannot be used in growth experiments due to the impermeability of the cell membrane toward them. The final answer to the question must await enzymatic experiments in whichznycelial barriers are eliminated. Dihydrouridine enhanced considerably the growth of the mold in the presence of small amounts of uridine and alpha~aminobutyric acid, while ureidoprOpionic acid riboside had no effect. The explanation for the great increase of growth in the presence of a small amount of 22 uridine or alpie-azzzinobutyric acid is not obvious. One possibility is that the compound is hydrolyzed.by the mold to dihydrouracil in the course of the growth on the uridine or aminobutyrate. Dihydrouracil (6) is known to stimulate the growth ‘of the mold in a fashion similar to that found.here for dinydrouridine. Another possible explanation is that once the moth is established on aminobutyric acid or on prop- ionic acid, the mold.may be able to produce in adaptive fashion an en- zyme capable of phosphorylating dihydrouridine. Alpha~aminobutyric acid and propionic acid prObably follow the same pathway in nucleic acid pyrimidine biosynthesis. This explains the identical lag phase in the growth of the mold on these coupounds (6) and the similar inhibition effect of arginine upon these compomdsdh) The obvious mthod of conversion of alpha~aminobutyric acid to prop- ionic acid would be the ole-animation of the amino acid, followed by oxidation, then decarbowlation. Alpha-ketobutyric acid would be an intermediate in this conversion. The finding that alpha-ketobutyric acid does support the growth of the mold at the pH of 3 while it did not promote the growth at the pH of )4 and of 5.6, indicates that alpha- ketobutyric acid is not able to penetrate through the cell membrane except in highly acidic medium. The conclusion of Boyd that the ketc- acid is not an intermediate in aeinobutyrate utilization is therefore not necessarily valid. ’ihis evidence mal-zes it likely that alpha-amino- butyric acid can be converted to propionic acid without passing through the intermediates homoserine and beta-hydroxypropidnic acid. Succinic acid and glycine were the other two compounds which were affected in their growth promoting action by the change of the pH of the medium. The pK values of propionic acid and succinic acid are h.87 :fi ‘ .’o~. 23 and b.19 respectively, therefore no great differences in the permeabil- ity would be expected on the basis of the pH of these compounds. The similarity in the growth promoting action at the pH of h of these com- pounds and the complete lack of growthspromoting ability of succinic acid at the normal pH would indicate that succinic acid is probably farther away from the nucleic acid pyrimidines in the biosynthetic pathway; therefore the presence of greater concentration of succinic acid in the cell would.be required before effective growth could take place. The growth with glycine cannot be explained at the present time. Presumably it can be converted relatively directly to a compound of the bioavnthetic path leading toward pyrimidines. .1 ...,_ _ Eur-3 l. 2. SUMMARY Experiments were carried out to obtain evidence concerning the nature of the alternate route for nucleic acid pyrimidine bio~ synthesis used hy the mutant.E, E£E§22.1298- Dihydrouridine and ureidopropionic acid riboside were synthesized. The mold was not able to utilize either of these compounds for growth when they were used as sole supplements to the basal nutrient medium. Dihydrouridine greatly enhanced the growth of the mold on uri~ dine and on aminobutyric acid. Ureido-propionic acid riboside had no effect upon the growth of the mold with these compounds. These experiments do not provide evidence in support of the bio~ synthetic pathway'postulated (6) for nucleic acid pyrimidine bio- synthesis from.propionic acid. The possibility exists, however, that the mold is not able to phosphorylate the nucleosides. Succinic acid and glycine were utilized at a.pH of h, but were not utilized at the normal pH of 5.6 or at a lower pH. Alpha-ketobutyric acid at a pH of 3 was utilized by the mold as well as was propionic acid, but at a pH of h and at a pH of 5.6 did not support the growth of the mold. The conversion of alpha-aminobutyric acid to prOpionic acid through the intermediate alpha—ketobutyric acid is suggested. 1. 10. 11. BIBLIOGW A. Kornberg, in W. D. McElroy and B. Glass, The Chemical BasLs of Heredity, Johns Hopkins Press, Baltimore, 1956. Finis, K., Henderson, R. B. and Fink, R. 11., J. Biol. Chem, 121, hhl (1932); egg, 3&9 (1953). {-3 . Canellakis, e. 3., J. Biol. Chem., 2e1 315 (1957); egg, 329 ~-*’ m I-‘airley, J. 1..., J. Biol. Chem, 312, 3147 (1951;). Herrmnn, R. L., and Fairley, J. 1..., J. Biol. Chem, £31, 1109 (1957). Boyd, J. PL, Unpublished Thesis, Michigan State University, (1958). Teas, H. J., Horowitz, N. H., and Fling, 14., J. Biol. Chem” 11%, 651 (19L8 . Davis, B. D., Advances in Enmnology, Vol 16, Nord, 1“. F. ed., Interscience Publishers, Inc. , 1955, p. 2&7. Moke‘asch, L. 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