STUD-£55 QN mmsaammmsg ENZYMES cw: NEURCSPQRA CRASSA 1298 Thesis for ”10 Degree of M. 5. IVHCHEQAN STATE UNIVERSITY D. Gene Wampler 1961 'I'HES'S 1! ‘ IL, :Jii‘; Michigan 51‘“ ‘ University j yn'rr ‘)..,“ EAB‘I LAr-ngu, ’."1:"~-’TPS!TY ‘ ,\.‘."..'~.'J SCILNCE MOUH!GAN ABSTRACT STUDIES ON TRANSCARBAMYLASE ENZYMES OF NEUROSPORA CRASSA 12.98 by D. Gene Wampler The presence and activity of some transcarbamylase enzymes in the pyrimidineless mold, Neurospora crassa 1298, were studied. These enzymes catalyse the general reaction: R-NHZ + NHz-CO-O-PO3: ——-> R-NH-CO-NHZ + HPO4: The presence of transcarbamylase activity in water soluble extracts of mold mycelia was detected by the Koritz-Cohen colorimetric test for the ureido group formed. The amount of protein in the enzyme preparation was determined colorimetrically by its reaction with Biuret reagent. Enzyme activity was then expressed in umoles of product formed per milligram of protein per hour of reaction time. It was found that when the mutant is grown on uridine the initial level of aspartic transcarbamylase activity is very low, and as the organism grows, this level of activity rises. When the organism is grown in a-aminobutyrate, aspartic transcarbamylase activity remains at a rather high level. I The level of ornithine transcarbamylase activity appears to be unaffected by either the type of nutrient supplied or the amount of growth. The activity of this enzyme is, however, affected by arginine and several aliphatic acids, including some which are known to promote growth of the organism in the absence of pyrimidines. STUDIES ON TRANSCARBAMYLASE ENZYMES OF NEUROSPORA CRASSA 1298 BY D. Gene Wampler A THESIS Submitted to Michigan State University . in partial fulfillment of the requirements for the degree of MAS TER OF SCIENCE Department of Chemistry 1961 A CKNOWLEDGMENTS The author expresses his thanks and appreciation to Dr. James L. Fairley for his guidance and assistance in making this thesis possible. »Appreciation is also expressed to Dr. Roland H. Davis for supplying cultures of the pyrimidineless mutants 45502, 63902, and 68902, and for his help in making the method of analysis workable. Gratitude is also due the National Institutes of Health for financial assistance during this project. >§< ::< :',< a}: 2:: :}< J}: >:: >:< >:< :1: 3:: ii To Theresa iii TABLE OF CONTENTS Page INTRODUCTION ........................ 1 MATERIALS AND METHODS ................. 6 Organisms ............. . .......... 6 Growth of Organisms .................. 6 Preparation of Acetone Powder ......... . . . 7 Preparation of Enzyme ................. 7 Enzyme Assay ................... 8 RESULTS ............................ 13 DISCUSSION .......................... 18 SUMMARY ........................... 22 REFERENCES ......................... 23 APPENDIX ....................... . . . . 24 iv LIS T OF TA BLES TABLE Page I. ASpartic Transcarbamylase and Ornithine Trans- carbamylase Activities from N. crassa 1298 . . . . 14 II. Effect of Several Aliphatic Acids on Ornithine Transcarbamylase from N. crassa 1298 . . . . . . 15 III. Relation of Growth Promoting and Inhibiting Power of Several Aliphatic Acids . . ......... l9 LIST OF FIGURES FIGURE Page I. Lieberman-Kornberg Pathway for Pyrimidine Synthesis ....... ..... 3 II. Fairley-Boyd Pathway for Pyrimidine Snythesis in N. crassa1298. ................. . . 4 III. Typical Assay for Transcarbamylase Activity. . . . 12 IV. ASpartic Transcarbamylase and Ornithine Trans- carbamylase Activities as a Function of Per Cent Maximum Growth ............. . . . . . .. 16 V. Apparent Inhibition of Ornithine Transcarbamylase . 17 vi IN TRODUC TION Neurospora has been a familiar pest to bakers for well over a hundred years as a red mold growing on bread. Around the turn of the century a Dutch botanist, Professor F. A. F. C. Went (1), found the natives of Java fermenting peanut meal with NeurOSpora to form a delicacy which they called "ontjomY'. Professor Went took the mold back to Holland and worked with it in his laboratory. In 1927 Shear and Dodge (2) published work they had done with the organism and proposed the names N. sitophilia, N. crassa, and N. tetraspora for the three species. In 1941 Beadle and Tatum (3) reported the production of mutant strains of N. crassa by treatment of the mold with X-rays. Mutant strains were identified by their inability to grow on a minimal medium containing only inorganic salts, an inorganic nitrogen source, biotin, and a simple organic carbon source such as sucrose, and their ability to grow when more complex organic molecules, such as pyrimidines or amino acids, were added. » Since 1941, mutations have been produced by treatment with X-rays, ultraviolet radiation, and neutron bombardment. Over 80, 000 single Spore strains have been isolated, of which some 500 are nutritional mutants. About 43 of these mutants required the pyrimidine, uracil, for growth and were named the pyrimidineless mutants. Genetic studies have made it possible to arrange these mutants into groups pyr 1, pyr2, pyr3, etc. The pyr 3 group contains a number of allelic mutants designated pyr 3a, 3b, 3c, and 3d. N. crassa 1298 (often referred to as 1298) is one of these pyr 3 mutants. There is some evidence that 1298 is a representative of the pyr 3a type, but such a position in the genetic map has not been definitely established. Normally a nutritional requirement is eXplained by assuming that some step along the biosynthetic pathway of this nutrient is blocked or deficient. The pathway of pyrimidine synthesis (Fig. I) which is generally accepted for Neurospora and which has been demonstrated to be present in numberous other organisms, was proposed by Lieberman and Kornberg (4). Early work with pyrimidineless mutants, therefore, centered around attempts to find intermediates of the Lieberman- Kornberg pathway which would support growth. No such intermediates were found. However, Loring and Pierce (5) did find that the pyrimidine nucleosides, such as uridine or cytidine, were much more effective in promoting growth than the free bases; Mitchell and Houlahan (6) found that the mutants will grow, although very poorly, on a few aliphatic acids such as oxalactic acid and aminofumaric acid. Fairley (7) has found that the mutant 1298 will grow on several simple aliphatic compounds, including a-aminobutyrate and sodium propionate. Furthermore, he and Boyd (8) have found that carbon-14 labeled a-aminobutyrate and propionate are incorporated rather specifically into the pyrimidines of the mycelial ribonucleic acids, although with considerable dilution; that when labeled uracil is fed to the mutant the radioactivity is appreciably diluted; that when grown on uracil there is a considerable lag period before growth begins. On the basis of this and other evidence they have proposed an alternate pathway of pyrimidine synthesis (Fig.- II). One striking feature about the growth of 1298 on a-aminobutyrate is that this growth is arrested‘by extremely small quantities of arginine. Fairley and Adams (9) have foundvthat as little as 0.04 pg of arginine per ml of nutrient will prevent growth. A similar case of arginine affecting the growth of a pyrimidineless mutant has been reported by Houlahan and Mitchell (10). They found that Figure I. Lieberman-Kornberg Pathway for Pyrimidine Synthesis COOH NH; CH. l + O=C CH | HZN/ \c OOH 0130;? aspartic acid carbamyl phosphate O >1< HN r l s O NH COOH orotic acid HN O I? COOH ribose-5'-P orotidine-S' - phosphate >’.< Indicates trans carbamylation step. COOH Nil H COOH ureido-succinic acid 0 HN O ;\ COOH NH dihydroorotic acid I ribose-S'eP uridine-5' - phosphate Figure 11. - Fairley-Boyd Pathway for Pyrimidine Snythesis in N. crassa 1298 COOH COOH I I COOH COC A NHz-CH O=C O I ———>- ' -———-—>- CH2 CH2 CH2 —""_>" (EH; I I I CH3 CH3 CH3 CH3 a-amino- a-keto- propionic propionyl- butyric acid butyric acid acid Coenzyme A COOH COCoA COSOA <|3Hz \clsnz <|3Hz CH2 < CH2 < CH2 Hli/ HN/ HZN/ ribose-5'-P \ribose-5'-P B-alanine B-alanyl-CoA B-alanyl- 5'-ribotide 5'-ribotide Coenzyme A >3 COOH O O HZN \ N )3 x N l i N 1? ribose-5'-P Il'ibose-5'-P ribose-5'-P N-carbamyl-B-alanine dihydrouridine- uridine-5'- 5'-ribotide 5' -phosphate phosphate 3:: Indicate s transcarbamylation step . pyr 3a (37301) will take a second mutation, s (suppressor), and that the resulting double mutant, pyr 3a—s will grow without pyrimidines. Addition of O. 5 moles of arginine per ml of medium will restore the pyrimidine requirement. Although no evidence has been presented that arginine directly affects enzymes of pyrimidine synthesis, Gorini and Maas (11) have shown that when Escherichia coli is cultured in the presence of arginine, the level of ornithine transcarbamylase is lower than normal. There are several other reasons to suspect a relationship among arginine, transcarbamylase reactions, and pyrimidine biosynthesis in 1298. Davis (12) has demonstrated that pyr 3d (45502) lacks the enzyme aspartic transcarbamylase. If the pyr 3 mutants are true alleles, this evidence would indicate that the pyrimidine requirement is at least related to aSpartic transcarbamylase in all of the pyr 3 mutants. This observation would then relate aspartic transcarbamylase to the arginine effect and therefore to ornithine transcarbamylase. Of course, aspartic transcarbamylase and ornithine transcarbamylase are already related through the common substrate, carbamyl phosphate. A further inter- relationship can be found in the data of Mitchell and Mitchell (13) which imply that the synthesis of arginine and pyrimidines is somewhat competitive. With the above information at hand it seemed advisable to study the quantitative relationship of aspartic transcarbamylase and ornithine transcarbamylase from Neurospora crassa 1298, and the possibility that some of the compounds which affect growth of the organism also affect these enzymes . MA TERIA LS AND ME THODS Organisms Mutant strains of Neurospora crassa used in this study were pro- duced by Beadle and Tatum (3). _l_2_9_§_ was part of the collection here at Michigan State University. It had been kept in the laboratory here for a number of years and had acquired the ability to grow slightly in basal-mediuml after a long induction period. Toward the end of the project a new culture was received from R. L. Herrmann who had receiVed it from the American Type Culture Collection in Washington. This new strain showed no growth after 20 days on basal medium and has been designated £2831. to avoid confusion with the original 1298. 45502, 63902, and_6_8_90_2were received from R. H. Davis from his collection at the University of Michigan. Growth of Organisms The organisms were maintained on 2% agar slants containing a supplement of 80 ug uridine per ml of agar. In order to keep growth of the organisms fresh, they were transplanted to new slants at least once a month. The molds were grown in 125 ml Erlenmeyer flasks stoppered with cotton plugs. Each flask contained 25 ml of basal medium and was supplemented with 2 mg of uridine (80 pig/ml) or with 5 mg of a-amino- butyric acid (200 ug/ml). The three media (basal, uridine, and a-amino- butyric acid) were made up in large quantities, autoclaved, and siphoned 1See Appendix for preparation of all reagents. out just before using. Flasks and contents were sterilized after filling, and allowed to cool before being incoculated. To inoculate the medium, a spore suspension was prepared by gathering a mass of mycelia and Spores on an inoculating needle and suSpending the Spores in 2-5 ml of sterile water. Each flask was inocu- lated with from 1,-3 drOps of the cloudy spore suSpension, the number of drops depending on the number of flasks to be inoculated. The mold was then allowed to grow at room temperature in the darkness ofa desk drawer. Preparation of Acetone Powder Mycelial pads were harvested by filtering the contents of the flasks through a Buchner funnel, washing quickly with about 20 m1 of water and then immediately with several 20 m1 portions of room-temperature acetone which had been dried over NazSO4. The water-free mycelial pads were then homogenized in cold (~50) dry acetone in a Vertis homogejniz'er.'__ This suspension was again filtered through a Buchner funnel, the powder cake allowed to air dry and stored at room temperature. The acetone powder remained stable for at least a month. Preparation of Enzyme Throughout the following preparation, care was taken to keep materials at a temperature of 00 or colder. Most of the work was done in an ice-salt bath having a temperature of about -30. About 100 mg of acetone powder was extracted with 6 ml of 0. 02 M tris-acetate buffer with the use of a small Potter tissue homogenizer. The suspension was centrifuged in a cold clinical centrifuge. The pre- cipitate was resuspended in an additional 3 m1 of buffer and reprecipitated in the cold centrifuge. The combined supernates were then centrifuged in a refrigerated ultracentrifuge at 13, 000 r.p.m. (20, 000xG) for 2-»4 minutes. This final supernate was used directly for the aspartic transcarbamylase assay, or diluted with 2 volumes of 0. 02 M tris-acetate buffer for the ornithine trans carbamylas e as say. 7 Enzyme Assay Developing an acceptable method of assaying for the presence of a transcarbamylase enzyme was a major problem. The method of Koritz and Cohen (14), used by Davis for his studies of transcarbamylase activity, was avoided at first because arginine was known to interfere, and one of the studies planned was to test aspartic transcarbamylase activity in the presence of arginine. As it turned out, passing the reaction product through a short column of Dowex- 50W removed the arginine. In the case of ornithine transcarbamylase, a blank containing arginine was also run and the amount of interfering color subtracted. Before this method was developed, an attempt was made to follow the reaction by assaying for the inorganic phosphate liberated, using the method of Lowry and Lopez (15). After several months of trying,‘ without success, to adapt this method to the assay at hand it was abandoned and the Koritz- Cohen method was again employed. One of the reasons that the Lowry- Lopez method did not work may be that carbamyl phOSphate is rather unstable, and, therefore, liberated a large background of inorganic phosphate. Returning to the Koritz-Cohen method did not immediately solve all of the problems. Several "tricks" in the method had to be mastered before the results were reproducible. Right up to this writing minor changes have been made in the procedure. The method to be described on pages ’9-10 is not the method by which all of the assays were run, but it is the method which seems now to work best. The more recent changes were, however, mostly fine points and should not make a significant difference in the results. For instance, in the first part of the work aSpartic acid was neutralized against a pH meter in 25 m1 quantities and kept for up to 10 days. Toward the end of the work, the acid was neutralized in 5-10 ml quantities just before using and the pH adjusted against pH paper. The most serious change was the change in the 1298 mutant, which has already been mentioned. However, data received from the two mutants did not differ significantly. For the aspartic transcarbamylase assay, the following reagents were mixed in an 8 inch test tube, in the order given, and allowed to react for 15 minutes at room temperature: 0. 25 m1 of 1.0 M glycine-NaOH buffer pH 9.1 l. 0 ml of potassium aspartate solution pH 9 1. 0 m1 of enzyme preparation 1. 0 ml of carbamyl phosphate solution At the end of 15 minutes the reaction was stopped by the addition of 0. 5 m1 of 2 N perchloric acid. The denatured protein was allowed to coagulate for a few minutes and then precipitated in a clinical centrifuge. A 3 m1 portion of the supernate was decanted and allowed to pass through a column containing 1 m1 of Dowex—SOW-hydrogen form. The column was washed with a 2 ml and then a 1 m1 portion of water, giving a final volume of 6 m1. To 2 ml of this final solution was added 4 ml of a 50% solution of? sulfuric acid, 0. 2 ml of diphenylamine-p-sulfonate, and 0. 2 m1 of diacetylmonoxime. The tube was capped, heated in a boiling water bath for 10 minutes and then cooled rapidly to room temperature. To the cooled solution was added 0. 2 ml of potassium persulfate and the solution returned for exactly one minute to the boiling water. The solution was then cooled once again and the color allowed to develop for 10 minutes away from direct sunlight. 10 After 10 minutes the optical density was read on a Klett-Summerson Photoelectric Colorimeter, using a green (540 mu) filter. The readings, kept in the range of 100-300 units, were converted into concentration of product by reference to a standard curve. The standard curve was pre- pared by adding known amounts of urido-succinate to water blanks and running them through the assay. At the same time that the assay for product was run, a 1 pr 2 ml sample of enzyme was diluted with water to 5 m1, and 5 m1 of Biuret reagent was added. After 20 to 40 minutes, the colorimeter was zeroed on a solution containing 5 m1 of Biuret reagent and 5 ml of water. Using the same green filter, the optical density of the protein-Biuret solution was read. This reading was converted to mg of protein by reference to a standard curve prepared with known amounts of egg albumin. For ornithine transcarbamylase, the following reagents were mixed in the order given: 0. 25 ml of 1.0 M tris-acetate buffer pH 9.1 1. 0 ml of ornithine solution pH 9 ml of enzyme preparation 00 2 . 8 ml of inhibitor or water 0 ml of carbamyl phosphate solution After 15 minutes the reaction was stOpped by the addition of 0. 5 m1 of 2 N perchloric acid. Since so little enzyme was used, the mixture was not centrifuged. Since the Dowex 50W column removes citrulline, the chromotagraphy step was omitted. Instead, 3 ml of the reaction mixture was taken and added directly to 3 ml of water. A 2 m1 portion of this diluted reaction mixture was then assayed as for aspartic transcarbamylase. In this case, however, the reference standard was citrulline rather than ureido-succinate. 11 When B-methyl aspartic acid and B-alanine were used as possible substrates for the reaction, the assay for aspartic transcarbamylase was used. Figure 111 shows a typical assay for transcarbamylase activity. Controls which were a standard part of the assay were: Tube 1 Water blank used to set the base reading of the instrunaent. Tube 2 ”Time O” blank, giving the amount of color produced in the absence of a reaction. This tube was particularly important in the arginine studies. Tube 10 Reference standard containing a known amount of the product. 12 .mmcHH000n 30HVH 0H“ 80.3 300qu H00u0Hd0H00 0.83 m0udeH GOHHHHEGH 0:00u0a 0f .0GHHHDMHH0 m0 con—0.5000000 35000902: 5?» 003de 9.00:: 0 55 0000.02: mwcHH000u 30va 005mm 60003800 003 c3000 05 CH @000 $30.00: HmmH 0:» 005 083 05. SOS 00.300005 0H0 03.235 000:8 £0va“ 0.83 mwGHH000u 05 £033 marine Hd>u0ucH 053 05 0» u0w0u 000.038 0£HN .033 H0nH ma mm .0 Ho £33089 mo mg m .m 00 mpcomm0uu00 mHNHH .333 30g mv H0 mchm0u .m 0>0w doflmumm0um 0§>NG0 05 mo HE N 5?? 003000.“ H0ude <2 o MN on mew. om me we NCOHHHQEGH «:00 n0nH o oo . mo . oH . HNH . 0H . 0N . mm . HOHHQHHHGH H0. 030005 ON. am. 3.. mm. ON. AN. NN. 2. 888“ 323.58 3381 mHHdm0m 0002300100 Hum mom :N owH NHNH HNH HoH cm 08H”. o 90m 0080.300 «a EN EN :2 NE NH NS S H 0 8033038 .58 S No wHN mmH :2 H o 000555 mHuNH pm Elm wHN 3: 2H NNH NH: .5 o 0 000.055 NHuw wachm0yH 30HVH x N x x x x x x x x 530.4 00£00nNHHuovH X X x x x x x x o x . H.OHUHiH HE m .o .erG mH 08H“ :0HH000HH o x x x on x x x x 0 00023093 3803000 HE H o o o o o o o o x o .0200 28 m .o o x X x x x x x x o H0E>NG0 HE N .o o o N . v . o . w . o o o o 2 «.N .o o o o o o o e . w . w . o H2 wHN .o 300 0HH>HSQOGH50u0 HE x o o o o o o o o C 0:33.33 SHE H H8 N .o 0 un un an uh um Uh um um O @GMJHwCHO 2 v.0 .0 H8 H x x x x x x N x x x .0303 HE mm .o wN N. s. e. N. o N. o o N 0:1:E muz0m00m 0H m. w n b m HN m N H u0nESZ 00.38 0.200: HHNN .NoH H030 0HH>00£OGHEMIU a0 GBOuw qumH 80.3 39300 0mmH>EmnumomcmuB 00HfiHCHO m0 GOHHHHHHHGH H000 0HH35£02HE30< 0mmH>Emnueumnmufi no“ >mmm< H0395. .HHH 0udenH RESULTS Both ornithine transcarbamylase and aspartic transcarbamylase activities were found in NeurOSpora crassa 1298. When the organism was grown on a-aminobutyric acid, the level of aSpartic transcarbamylase activity remained consistently high, but when grown on uridine the level of activity started low and rose as the mold grew. The level of ornithine transcarbamylase activity seemed to be independent of the age of the mold. These data are given in Table I and are represented graphically in Figure IV. A preliminary study was made to determine whether or not arginine affected the activity of aspartic transcarbamylase. No effect was found. However, the concentration of arginine used in the study (2. l prnoles per tube) was much lower than that used to produce inhibition of ornithine transcarbamylase (190 umoles per tube). It was found that arginine slightly suppressed the formation of ureido groups by ornithine transcarbamylase. It was also found that a number 'of aliphatic acids, some of which were known to promote growth of the mutant, suppressed the apparent activity of ornithine trans;- carbamylase. One such compound raised the apparent activity. These data are recorded in Table II and in Figure V. (Figure V emphasizes interestingirelatioriiships'among the seven compounds tested. They seem (to fall into three rather distinct groups; two inhibit rather strongly, four rather weakly, and one increases the apparent activity. When 8- methylaspartic acid and 8-alanine were supplied as possible substrates for transcarbamylation, no reactions were detected. Mutants 45502, 63902, and 68902 did not grow on minimal medium supplemented with d—aminobutyric acid. 13 14 Table I. Aspartic Transcarbamylase and Ornithine Transcarbamylase Activities from N. crassa 1298 Growth Age in Weight % Maximum ATC * Medium Mutant Hours in mgs. Growth Activity a - Aminobutyri c acid 1298 66 -. 4 11 5. 5 " 1298 133 25 71 4.0 " 1298 133 25 71 3.9 " 1298 207 34 97 5.0 ” 1298H 113 ll 22 5.8 ” 1298H 113 11 22 6.4 Uridine 1298 43 10 21 1.0 " 1298 43 10 21 2.6 " 1298 66 37 75 3. 3 " 1298 66 37 75 4.0 " 1298 133 46 92 2.9 " 1298 133 46 92 3.0 ” 1298 207 44 89 3. 8 Uridine 1298H 27 2.8 3.7 0.27 " 1298H 40 20 27 1. 0 OTC Activity):< Uridine 1298H 40 20 27 5. 8 o-Aminobutyric acid 1298H 241 100 6.0 ATO = Aspartic Transcarbamylase OTC = Ornithine Transcarbamylase :5: Activity is expressed in moles of product formed per mg of protein per hour of reaction time. 15 Table II. Effect of Several Aliphatic Acids on Ornithine Transcarbamylase from 1_\I_. crassa 1298 mM bf OTC OTC Activity):< ' % of Inhibitor Inhibitor Activity* with Inhibitor . Control a-Aminobutyric acid . 12 6. 0 3. 6 60 Arginine .19 5. 3 88 a—Aminobutyric acid . 05 6. 2 4. 4 78 " .10 4. O 64 " .14 3. 2 51 " . 19 2. 7 44 " . 23 2. 3 37 " . 38 2. 0 32 - Homoserine . 32 6.7 4.7 71 " . 12 6. 2 93 Threonine . 32 5. 7 85 " . 12 6.1 91 2, 4-Diaminobutyrate . 32 2. 9 44 " .12 4. 6 69 Propionate . 04 5. 3 5. 5 104 " . 12 5. 7 109 ” . 24 6. 0 115 " . 32 6. 1 116 Alanine . 24 4. 4 83 " . 08 5. 0 94 All data taken from 1298H grown on a-aminobutyric acid for 241 hours, 100% maximum growth. Ornithine concentration is 0.04 mmoles per tube. OTC = Ornithine Transcarbamylase *Activity is expressed in moles of product formed per mg of protein per hour of reaction time. 16 Figure IV. Aspartic Transcarbamylase and Ornithine Trans- carbamylase Activities as a Function of Per Cent Maximum Growth 0 I I I I I I I I I 1 10 20 30 40 50 60 70 80 90 100 % Maximum Growth A Spartic Trans carbamylas e o 1298 o 1298H -- grown on a-aminobutyrate A 1298 A 1298H -- grown on uridine Ornithine Transcarbamylase D 1298H grown on a-aminobutyrate I 1298H grown on uridine Figure V. Apparent Inhibition of Ornithine Transcarbamylase 70 —— x 60 — o/ 50 — 40 — G 0 fl .1: 20 — s fife—r. E 10 " '/ a) 0 ‘ D o i-I o 04 -10 _" ‘\ 4H.“ '20 I I I I 0 0.10 0.20 0.30 0.40 mmoles of Inhibitor per Tube x = o-aminobutyrate 0 = threonine o = 2, 4-diaminobutyrate O = alanine A = homoserine I = propionate A = arginine Ornithine concentration is O. 04 mmoles per tube. DISCUSSION This study has demonstrated the presence of aspartic transcarbamy- lase activity in Neurospora crassa 1298, when grown on both a-amino- butyric acid and on uridine. It has also been shown that pyr 3d (45502) and two other pyr 3 mutants (63902 and 68902) will not grow on basal medium supplemented with u-aminobutyric acid. This information, coupled with the finding of Davis (12) that the pyr 3d mutant has no aspartic transcarbamylase, suggests that 1298 is not a pyr 3d mutant. However, since 1298 is still in the pyr 3 group, the basis for the pyrimidine requirement must still be connected somehow with the aspartic transcarbamylase enzyme. While some of the known transcarbamylase reactions were being run, it was decided to try a few compounds as possible substrates for unknown transcarbamylation reactions. One of the substrates for trans- carbamylation in the Fairly- Boyd pathway is fi-alanine. This compound did not produce detectable amounts of the ureido function under the con- ditions of the assay employed. Another compound tested was [SI-methyl- aspartate. This was tried mainly to test the specificity of aspartic transcarbamylase, but also because of the possibility that such are.- action might play a role in the biosynthesis of thymine. Like fi-alanine, B-methylaspartate gave no reaction. Although neither compound gave positive results, it cannot be con— cluded that their transcarbamylases are not present in the mycelial extract. Possibly something in the assay system inhibits the reaction, as glycine in the glycine-NaOH buffer inhibits ornithine transcarbamylase. Or perhaps the reaction is favored only at a pH more nearly that found in living myc elia . 18 l9 Yates and Pardee (16, 17) have found that pyrimidine synthesis in E. coli is normally controlled by controlling the aspartic transcarbamy- last reaction. This reaction is controlled by pyrimidines. Thus pyrimidines control their own synthesis. The results obtained when 1298 was grown on uridine suggest a repression mechanism very similar to that found by Yates and Pardee in E. (£113; During early stages of growth, when there is abundant uridine present, the level of aspartic transcarbamylase is very low. As the mold grows, presumably using up the fed uridine, the level of transcarbamylase rises. These data, suggesting repression, are further evidence that the mutation in 1298 is not simply an insufficient amount of aspartic trans- carbamylase. If this were the case, the mold should grow best when the aspartic transcarbamylase level is the highest, that is, on the least amount of uridine. In fact, however, increasing uridine concentration increases growth rate. The ability of the mold to control the level of aSpartic trans- carbamylase appears to be normal. Of course, this does not mean that 191113 activity of the enzyme is normal. It might easily be the case that within the cell the enzyme is inhibited. There is reason to believe that arginine may be such an inhibitor. The fact that when the organism is grown on fi-aminobutyric acid the level of aspartic transcarbamylase is higher than is found (12) in the wild strain (about 4. 5 pM /mg /hr as compared with 1.44 pM /mg /hr) might suggest that this acid stimulates production of the enzyme. However, it is more likely that this high level of enzyme is due to the deficiency of uridine (or other pyrimidine), since levels of aspartic transcarbamylase as high as 13 uM/ mg/ hr have been obtained (18) when the organism is grown on limiting concentration of uridine. The discovery that arginine exerts a powerful inhibition- on the growth of 1298 seems to be an important step in the attempt to explain 20 the pyrimidine requirement of this mutant. ' Evidence which suggests a relationship between arginine and pyrimidine synthesis was presented in the introduction. If arginine does in some way prevent the synthesis of pyrimidines, the logical way to produce growth would be to remove arginine. Since ornithine transcarbamylase is essential for the pro- duction of arginine, a comparison of the ability of a compound to inhibit ornithine transcarbamylase and its ability to promote growth might be useful. - Such a comparison is given in Table III: Table III. Relation of Growth Promoting and Inhibiting Power of Several Aliphatic Acids Growth Acid in mg1 ‘70 InhibitionZ a-Aminobutyric acid 43 57 Propionic acid 22 -113 Homoserine 18 18 Threonine ll 13 - 2, 4-Diaminobutyric acid 5 44 Alanine 0 l4 1Data from Fairley, e_t a_._l. (19). zData from Table II, this paper. 3A minus. number indicates that enzyme activity was increased rather than inhibited. Growth is expressed in dry weight of mycelia produced in 4 days when 0. 05 mmoles of the acid is added to 25 ml of basal medium (19). Inhibition is the per cent decrease in activity caused by the addition of 0. 2 mmoles of the acid (5 times the ornithine concentration) to the o rnitine trans carbamylas e as s ay. 21 The data presented in Table 111 does not seem to indicate a positive correlation between the ability of a compound to promote growth of 1298 and its ability to inhibit ornithine transcarbamylase. However, it must be remembered that the enzyme preparation used was only a crude extract, and many reactions may be taking place other than the one being studied. The data may reflect the ability of the "inhibitors" to undergo transcarbamylation reactions of their own. . For instance, threonine might be inhibiting ornithine transcarbamylase strongly, and, at the same time be undergoing a transcarbamylation reaction of its own. The net formation of the ureido function would then be the sum of these two opposing factors. It is also possible that a compound added as an inhibitor might be rapidly converted into something else which could undergo a transcarbamylation reaction. For instance, propionate, which cannot undergo a "propionic transcarbamylation" reaction, might be converted into something else which could act as a carbamyl acceptor. If, however, the data are taken at face value it appears that the a-amino acids generally act as inhibitors and that similarity of structure to ornithine is an important requirement. On the other hand, if struc- tural similarity is the key, it is difficult to understand why a-amino- butyrate is a better inhibitor than 2, 4-diaminobutyrate. The data presented in this study, together with previous work on the ability of arginine to suppress growth of the mutant studied, suggest the possibility that the pyrimidine requirement in 1298 is closely connected with the presence of arginine. They suggest further that the removal of this requirement by a-aminobutyric acid and other aliphatic acids may be effected by limiting the amount of arginine present, through controlling the activity of ornithine transcarbamylase. SUMMARY The levels of aspartic transcarbamylase and ornithine trans- carbamylase activities in NeurOSpora crassa 1298 were studied. The presence of these enzymes was detected by allowing aqueous extracts of mycelia to catalyze the production of ureido-compounds. The product of this reaction was detected colorimetrically by the method of Koritz and Cohen. It was found that the level of aspartic transcarbamylase activity varied with the medium in which the organism was grown and in some cases with the stage of growth. Ornithine transcarbamylase was also found to be present in 1298. A number of aliphatic acids were found with the ability to inhibit this enzyme. One acid was found which apparently increased its activity. Attempts to identify other transcarbamylase enzymes in the organism were unsuccessful. The mutants 45502, 63902, and 68902 were found to be unable to grow on basal medium supplemented with u-amino- butyric acid. The implications of these findings as they relate to pyrimidine synthesis in 1298 are discussed. 22 -J 10. 11. 12. 13. 14. 15. l6. 17. 18. 19. .- Mitchell, H. K. , and Mary B. Houlahan, Federation Proc. , 6, REFERENCES . Went, F. A. F. C., Centralb° f. Bacteriol., Abteilung 2, 1, 544 (1901). . Shear, C. L., and B. O. Dodge, J. Agric. Res., _3_4_, 1019 (1927). . Beadle, G. W., and E. L. Tatum, Proc. Natl. Acad. Sci., _21 499 (1941). . Lieberman, 1., and A. Kornberg, J. Biol. Chem., 212, 909 (1955). . Loring, H. S., and J. G. Pierce, J. Biol. Chem., 1 3, 61 (1944). 506 (1947). . Fairley, J. L., J. Biol. Chem., 210, 347 (1954). Boyd, J. B., and J. L. Fairley, J. Biol. Chem., 234, 3232 (1959). Fairley, J. L., and A. B. Adams, Science, in press. Houlahan, Mary B., and H. K. Mitchell, Proc. Natl. Acad. Sci., 33, 223 (1947). Gorini, L., and W. K. Maas, Biochim. et BiOphys. Acta, 2_5,_ 208 (1957). Davis, R. H., Federation Proc., _4_6, 677 (1960). Mitchell, M. B., and H. K. Mitchell, Proc. Natl. Acad. Sci., E, 205 (1952). Koritz, S. B., and P. P. Cohen, J. Biol. Chem., 209, 145 (1954). Lowry, O. H., and J. A. Lopez, J. Biol. Chem., 162, 421 (1946). Yates, R. A., and A. B. Pardee, J. Biol. Chem., 221, 757 (1956). Yates, R. A.,'and A. B. Pardee, J. Biol. Chem., 227, 677 (1957). Davis, R. H., Personal Communication. Fairley, J. L., R. L. Herrmann, and J. M. Boyd, J. Biol. Chem., 234, 3229 (1959). 23 A PPE NDIX PREPARATION OF SOLUTIONS 24 REA GENTS General 0.02 M Tris-acetate buffer - pH 8.0 Dissolve l. 21 g of Tris (Tris (hydroxy-methyl) aminomethane) (Sigma Chemical Corp.) in 400 ml water and adjust to pH 8. 0 with acetic acid. Dilute to 500 ml. 1.0 M Tris-acetate buffer - pH 9.1 Dissolve 12.114 g of Tris in 80 ml water and adjust to pH 9.1 with acetic acid. Dilute to 100 m1. 1. 0 M Glycine-NaOH buffer - pH 9. 1 Dissolve 7. 505 g of Glycine and 5.845 g NaCl in 70 ml water and add strong (15 N) NaOH to pH 9. 1. Dilute to 100 ml. 2 N Perchloric acid Dilute from concentrated (ll. 8 N) perchloric acid. Biuret Reagent Dissolve 0. 75 g c0pper sulfate and 3.00 g sodium potassium tartrate in 150 ml 10% sodium hydroxide and make up to 500 ml with COz-free water. Dowex 50W-X12 Dowex 50W-X12 100-200 mesh (Dow Chemical Co.) is washed with water, NaOH, water till neutral, HCl, and finally with water again until neutral . Reactants The following reagents should be made up just before using: 25 26 0. 04 M Potassium-L-aSpartate SuSpend 26. 6 mg of L—aspartic acid (Mann Res. Labs.) in 4 ml of water and neutralize with weak KOH to a pH of 9, as indi- cated by pH paper. Dilute to 5 ml. 0. 04 M Ornithine Adjust 33. 7 mg DL-ornithine hydrochloride (Nutritional Biochemicals Corp. , hereafter abbreviated Nut. Bio. Corp.) to pH 9 as above and make up to 5 ml. 0. 04 M Potassium-DL-B—methyl aspartate Adjust 29.4 mg of DL-fi-methylaspartic acid (Nut. Bio. Corp.) to pH 9 as above and make up to 5 ml. 0. 04 M B-Alanine Adjust 17.6 mg gof B-alanine (Pfanstiehl Chem. Co) to pH 9 as above and make up to 5 ml. 0. 01 M Carbamyl Phosphate Dissolve 15. 3 mg of carbamyl phOSphate (California Corp. for Biochemical Research) in 10 ml of water. ”Inhibitor s " All of the following solutions were made 0.4 molar and adjusted to approximately pH 9 with weak KOH. _. D,L—Alanine - (Nut. Bio. Corp.) 178 mg/5 m1 D, L-a-Amino-n-butyrate - 206 mg/5 ml L-Arginine - (Mann Res. Labs. ) 348 mg/5 m1 2,4-Diaminobutyrate - (Nut. Bio. Corp.) 168 mg/5 ml D, L-Homoserine - (Nut. Bio. Corp.) 238 mg/5 ml 27 Sodium Propionate - (Prepared by A. B. Adams in this laboratory from propionic acid from Eastman Kodak Co.) 192 mg/5 ml. D, L-Threonine - (Bios. Labs. Inc.) 238 mg/5 ml Assay 0.4%Diphenylamine-p-sulfonate - (Eastman Kodak Co.) 20 mg/5 ml 3% Diacetylmonoxime - 150 mg/5 ml 1% Potassium Persulfate - 50 mg/5 ml 28 COMPOSITION OF THE BASAL MEDIUM Grams per Liter Ammonium tartrate .............. 5. 0 Ammonium nitrate ..... A . . . . . . . . . l. 0 Potassium dihydrogen phosphate . . ..... l. 0 Magnesium sulfate .............. 0. 5 Calcium chloride ............... 0. 1 Sodium chloride ............ . . . . O. l Sucrose . . . . .......... . . . . . . 10.0 Biotin ...... . . (pg/liter) ..... . . 5.0 (Nut. Biol. Corp.) Trace Elements pg per liter Sodium tetraborate ......... . . 88 Magnesium 11 chloride ....... . . 45 Ammonium molybdate ......... 64 Copper 11 chloride . . ....... . . 270 Iron III chloride ............. 500 Zinc sulfate °7HZO ..... . . . . . 2000 Supplements pg per ml Uridine .............. . ...... 80 (Schwartz Labs 81 Nut. Bio.) a-Aminobutyric acid .............. 200 uAYZg'” LHLI‘MSMY LIdRARI