EFFECTS OF ASPARTATE ON GROWTH AND ON THE SYNTHESIS OF a-amylase IN PSEUDOMONAS SACCHAROPHILA

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1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp June, 1964 Copyright X 1964 by the American Society for Microbiology Printed in U.S.A. EFFECTS OF ASPARTATE ON GROWTH AND ON THE SYNTHESIS OF a-amylase IN PSEUDOMONAS SACCHAROPHILA JEROME M. EISENSTADTI AND HAROLD P. KLEIN2 Department of Biology, Brandeis University, Waltham, Massachusetts Received for publication 6 January 1964 ABSTRACT EISENSTADT, JEROME M. (Brandeis University, Waltham, Mass.), AND HAROLD P. KLEIN. Effects of aspartate on growth and on the synthesis of a-amylase in Pseudomonas saccharophila. J. Bacteriol. 87: Protein synthesis by starch-grown cells of Pseudomonas saccharophila is inhibited by low concentrations of D-aspartate, whereas assimilation continues for several hours under these conditions. These effects are also observed when the cells are grown with cellobiose or maltose, but not with a wide variety of other carbon sources. L-Aspartate at a ratio of about 6:1 completely reverses these effects, whereas adenosine partially does so. One possible basis of the inhibitory effects of D-aspartate is in the synthesis of adenosine from inosine. and for its purification were reported previously (Markovitz, Klein, and Fischer, 1956). Chemical determinations. Proteins were determined by the Folin-Ciocalteau reagent with the procedures of Lowry (Lane, 1957). This method was standardized against weighed dry samples of protein derived from P. saccharophila. The methods of Hurlbert et al. (1954) were used in the separation of nucleotides, and of Lieberman (1956) to assay for adenylosuccinase. Preparation of cell-free extracts. Two procedures were used to obtain active extracts. In one, 18- to 24-hr-old cultures (25 ml) of cells were centrifuged and washed twice with 2-ml samples of.33 M phosphate buffer (ph 6.8). The weighed, packed cells were ground with alumina (Alcoa, no. 31), after which the mixture was extracted with 5.5 ml of cold.5 M phosphate buffer (ph 6.8) per gram of cells. The resulting suspension was centrifuged in the cold at 25, X g for 3 min. The other method utilized sonic disruption. In this procedure, cultures, 18- to 24-hr old (5 ml), were centrifuged, and were washed twice with 2-mil samples of phosphate buffer; During investigations of the amino acid pool of Pseudomonas saccharophila (Markovitz and Klein, 1955 a), and of the effects of amino acids on the formation of a-amylase in resting and growing cells, it was observed that the addition of aspartate resulted in drastic inhibition of cellular protein synthesis. Brief reports on this phenomenon have been presented (Eisenstadt, Grossman, and Klein, 1959, 196), and it is the approximately 1 to 2 g (wet weight) of cells were resuspended in 1 ml of cold buffer. This suspension was treated in a Raytheon 1 KC sonic purpose of this communication to give a fuller account of oscillator for 1 min, and was then centrifuged these findings. at 25, X g for 3 min in the cold. The resulting MATERIALS AND METHODS supernatant fluid was clear and opalescent, and, The organism used in these studies, P. saccharophila, and the conditions used in its cultiva- above, was considerably less viscous. in contrast to the grinding techniques described tion, were described earlier (Eisenstadt and Klein, Chemicals. Adenosine triphosphate (ATP), 1959). In general, the procedures of Markovitz guanosine triphosphate (GTP), and inosine and Klein (1955 a, b) were used for induction of monophosphate (IMP) were obtained from the a-amylase and the preparation of resting-cell suspensions. Procedures for the assay of a-amylase Pabst Brewing Co., Milwaukee, Wis. All other chemicals were of chemically pure grade. 1 Present address: Department of Microbiology, RESULTS School of Medicine, Yale University, New Haven, Conn. Effects of aspartate on starch-grown cells. Figure 2 Present address: Life Sciences, Ames Research 1 illustrates the characteristics of the aspartate Center, NASA, Moffett Field, Calif. effect on starch-grown cells. In this experiment, 1355 Downloaded from on April 25, 218 by guest

2 1356 EISENSTADT AND) KLEIN1BJ. BACTERIOL. formation is inhibited approximately 65%, after an initial period of enzyme appearance. Ejffect of DL-aspartate concentration on inhibition of protein synthesis. The results of two experiments to determine the concentration of aspartic acid necessary to inhibit protein synthesis are shown in Table 1. When DL-aspartate is added to cultures at 3 various stages of the growth cycle, results of the TIME (HOURS) 25 ; TABLE 1. Inhibition of growth of Pseudomonas C13 saccharophila by DL-aspartatea 2 -< X - 5C 15 rn 1 M, en_ FIG. 1. Effect of DL-aspartate on protein and ca-amylase synthesis, and on turbidity of starchgrown cells of Pseudonmonas saccharophila. Cells from 5 ml of medium were harvested, washed twice with 2 ml of.33 M phosphate buffer (ph 6.8), and samples of this suspension were distributed into 25-ml flasks containing 5 ml of complete medium. was added to some of the flasks which were incubated for 3 min, after which (at zero time) starch was added to a final concentration of.2% to all flasks. At the intervals indicated, samples were removed for analyses. The broken lines indicate results obtained with the control culture, and the solid lines indicate the culture containing.1% DL-aspartate. Symbols: open circles, total protein; closed circles, turbidity; triangles, a-amylase in supernatant fluid. portions of a 15-hr-old culture were dispensed in complete medium without starch. was added to some flasks and, after 3 min of incubation, starch was added to all flasks. As is clear from the data, aspartate inhibits protein synthesis completely. However, it is evident that the turbidity of the cultures increases for several hours under these conditions. The presence of DL-aspartate, therefore, does not interfere with assimilation (Whelton and Doudoroff, 1945; Wiame and Doudoroff, 1951) during the initial growth period. Between the second and fourth hours, assimilation is reduced, and, after the fourth hour, it is completely inhibited. Amylase 5 Expt 1 2 Addition (.1%) (.1%) (.1%) (.1%) (.2%) (.1%) (.7%) (.5%) D)L-Aspartate (.3%) L-Aspartate (.1%) Change in culture turbidity at 54 m,ub Change in cellular protein (Jgsg/O.l ml)c Amylase activity (units/ml)d a Starch-grown cells (5 ml) were harvested, washed twice with.33 M phosphate buffer (ph 6.8), and resuspended in 25 ml of complete medium with the indicated additions. They were incubated for 3 min at 3 C, with shaking, after which starch was added to a final concentration of.2%, and the incubation continued for an additional 6 hr. Samples were removed for culture turbiditv measurement and protein and amylase activity measurements. b Initial culture turbidity was approximately 15 in experiment 1, and 13 in experiment 2. Figures refer to Klett readings with a green filter. c Initial protein was 2 to 21,ug/.1 ml in experiment 1 and 17 to 18 Ag/ml in experiment 2. d In experiment 1 no amylase assays were performed. Downloaded from on April 25, 218 by guest

3 VOL. 87, 1964 ASPARTATE INHIBITIONS IN P. SACCHAROPHILA 1357 type shown in Fig. 2 are obtained. A lag of approximately 2 hr occurs before a marked change in the rate of protein synthesis takes place. Furthermore, at a time when the culture is in the logarithmic phase of growth, addition of DLaspartate shows no inhibitory effects. It is interesting to note that the effect of aspartate appears to change the rate of protein synthesis from a logarithmic rate to one that is linear. The possible implications of this will be discussed later. Effects of D and L isomers of aspartate. To determine which optical isomer of aspartate was responsible for the effects observed, a 25-ml culture of starch-grown cells was washed twice with buffer and resuspended in 1 ml of buffer. Samples of this suspension were incubated in 5 ml of complete medium containing either D-, L-, or DL-aspartate. After 6 hr, the results shown in Table 2 were obtained. Clearly, it is the D isomer that is responsible for the inhibition of protein synthesis. Indeed, the L isomer stimulates all of the synthetic activities of the cells that have been measured (Table 1). Effect of D-aspartate on cells not grown in starch. The results of investigations of the sensitivity to D-aspartate of cells grown in a variety of substrates are shown in Table 3. Of the substrates tested, the only ones yielding cells which exhibited sensitivity to D-aspartate at final concentrations up to.5% are starch, maltose, and cellobiose. [It is interesting to note that, of many substrates tested as inducers of the intracellular f3-amylase of this organism, only these three carbohydrates were effective (Thayer, 1953).] At higher concentrations, partial inhibition of protein synthesis could be obtained. Thus, with sucrose-grown cells, it was found that.1% D-aspartate inhibited protein formation by about 5%. That sucrose-grown cells differ from starchgrown cells is evident also from the fact that the addition of L-aspartate to the former did not stimulate either protein synthesis or culture turbidity. [In this connection, in experiments with nitrogen-starved cells (Eisenstadt and Klein, 1961), D-aspartate was found to be ineffective in replenishing the amino acid pool, and such cells could not be induced under resting-cell conditions.] Reversal of D-aspartate effects. Experiments to determine whether the D-aspartate effect on starch-grown cells could be reduced or reversed by the addition of casein hydrolysate indicated that a reversal of approximately 5% is possible with concentrations of casein hydrolysate of the order of.1 %. The ability of L-aspartate to reverse the inhibitions due to the presence of D-aspartate is illustrated in Table 4. A series of experiments of this type indicated that a reversal of the inhibitions by D-aspartate is in direct relationship to the amount of L-aspartate added, E CP 61 z jm 4 a. F- 2C TIME (HOURS) * -3 4 A~t-2 3 ~~~~A-1I XOi ~~~~- FIG. 2. Effect of addition of DL-aspartate at different stages of growth. The conditions are identical to those described in Table 1, with the exception that.1% DL-aspartate was added to individual cultures at the intervals indicated by arrows. The numbered curves correspond to the numbered arrows, which indicate the time of addition of DL-aspartate. TABLE 2. Protein synthesis by Pseudomonas saccharophila in the presence of D-, L-, and DL-aspartate* Change in Change in Amls Addition culture cellular amylasey at 54 mist (ug/.1 m)tunt/) Addition ] turbidity protein a(utiits/ml) L-Aspartate (.1%) D-Aspartate (.1%) D-Aspartate (.1%) (.1%) * The conditions of this experiment are similar to those cited in Table 1. t The initial culture turbidity was approximately 125 to The initial cellular protein content was 17 to 18,g/.1 ml. Downloaded from on April 25, 218 by guest

4 1358 EISENSTADT AND KLEIN J. BACTERIOL. TABLE 3. Effect of D- and DL-aspartate on cells of Pseudomonas saccharophila growing on various substrates* Change in cellular Growth substrate Addition protein Starch Sucrose Lactate Galactose Glucose Xylose Maltose Cellobiose Arabinose Acetate Melibiose Raffinose Mannose (A&g/.l Ml) Change in culture turbidity at 54 my * Cultures (1 ml) grown on the substrate indicated were harvested, washed twice in.33 M phosphate buffer (ph 6.8), and samples were resuspended in complete medium to obtain a Klett reading of 125 to 15. These suspensions, with the supplements indicated, were incubated for 3 min at 3 C with shaking (zero time). The original substrate was then added to a final concentration of.2%, and the incubation continued for an additional 6 hr. Samples were removed for culture turbidity and protein determinations, initially, and after 6 hr Downloaded from on April 25, 218 by guest a ratio of L- to D-aspartate of approximately 6:1 constant throughout the period of incubation being necessary for complete reversal. This over a 6-hr interval. reversal is reflected in culture turbidity, protein Because L-aspartate plays a central role in the synthesis, and amylase formation. Furthermore, synthesis of purine and pyrimidine bases (Lieberthe kinetics of the reversal were found to be man, 1956; Lukens and Buchanan, 1957), in

5 VOL. 87, 1964 ASPARTATE INHIBITIONS IN P. SACCHAROPHILA 1359 addition to its role in protein formation, selected purines and pyrimidines, or derivatives thereof, were tested for their ability to reverse the D- aspartate effects. Adenine, adenosine, inosine, guanosine, cytidine, uridine, and orotic acid were used in these experiments (Table 5). Of the compounds examined, adenosine was the only one to effect any reversal of the inhibition. This compound reversed the inhibition of protein synthesis to approximately 25 to 4% of the control. Because adenosine was found partially to reverse the D-aspartic effect, and L-aspartate could completely reverse the inhibition, it was of interest to find whether adenosine had any sparing effect on the amount of i-aspartate needed to reverse the inhibition caused by a given amount of D-aspartate. The results showed that adenosine exerts a sparing effect (Fig. 3). For example, concentrations of.3,.7, and.1% of L-aspartate reverse the inhibition by 32, 43, and 51 %, respectively. In comparison, similar concentrations of L-aspartate, with the addition of 5,g of adenosine, reverse inhibition by 54, 66, and 78%, respectively, whereas adenosine alone reverses the effect by 42%. It should be noted that the combined reversal by L-aspartate and adenosine is not additive. Because inosine was ineffective, and adenosine was moderately effective in reversing the D- aspartate inhibition of protein synthesis, a possible site of action of the D-aspartate may be assumed to be localized in purine synthesis in the reaction sequence: IMP-5' + L-aspartate + GTP -- adenosine monophosphate (AMP)-5' + fumaric acid + guanosine diphosphate + Pi To test this, cell-free extracts of starch-grown cells were prepared and assayed for adenylosuccinase. Under these conditions, L- but not D- aspartate was able to participate in the reaction (Table 6). To favor the accumulation of products, the reaction was allowed to proceed for a longer period of time, after which they were subjected to column chromatography on Dowex-1, and to paper electrophoresis (Black, Durrum, and Zweig, 1958). AMP is formed only in the presence of L-aspartate (Fig. 4 and 5). Because these experiments indicated the involvement of L-aspartate in the synthesis of AMP in P. saccharophila extracts, experiments TABLE 4. Reversal of D-aspartate growth inhibition by L-aspartate* Addition.1% DL-aspartate.1% DL- and.3% L-aspartate....1% DL- and.7% L-aspartate.1% DL- and.1% L-aspartate.1% DL- and.2% L-aspartate.1% DL- and.4% L-aspartate Increase in turbidity at 54 mut Increase in protein (JAgIO.1 Ml)T Amylase activity (units/ml) * The conditions of this experiment are similar to those cited in Table 1. t Initial culture turbidity was approximately 125 to 13. t Initial cellular protein content was 17 to 18 JAg/.1 ml. TABLE 5. Ability of various purines and pyrimidines to reverse inhibition by D-aspartatea Increase in Increase in Additionb turbidity protein at 54 mae (Ag/.l ml)d D-Aspartate D-Aspartate and adenine D-Aspartate and adenosine D-Aspartate and inosine D-Aspartate and guanosine D-Aspartate and cytidine D-Aspartate and uridine D-Aspartate and orotic acid a The conditions of this experiment are identical to those cited in Table 1. b D-Aspartate was added at.2%; all other additions were 5,ug/ml. c The initial culture turbidity was approximately 11 to 115. d The initial protein content was approximately 16 to 17,ug/.1 ml. Downloaded from on April 25, 218 by guest

6 136 EISENSTADT AND KLEIN J. BACTERIOLF E z m J- r 4I- 4 3C 2[ 4 3 -I _9~ ~ -6 -% u- ~o O- Z 1ot - o-i ~ ~ - I TIME (HOURS) FIG. 3. Sparing effect of adenosine on the reversal of DL-aspartate inhibition of protein synthesis by L-aspartate. The conditions are similar to those described in Table 1. The numbered curves correspond to the following incubation conditions: 1, control; 2, +.1% DL-aspartate; 3, +.1% DL-aspartate +.3% L-aspartate; 4, +.1% DL-aspartate +.7% L-aspartate; 5, +.1% DL-aspartate +.1% L-aspartate; 6, +.1% DL-aspartate + 5 fiag/ml of adenosine; 7, +.1% DL-aspartate +.3% L-aspartate + 5,ug/ml of adenosine; 8, +.1% DL-aspartate +.7% L-aspartate + 5,Ag/ml of adenosine; 9, +.1% DL-aspartate +.1% L-aspartate + 5,Ag/ml of adenosine. were performed to test the effect of D-aspartate on the formation of AMP with varying concentrations of L-aspartate. A Lineweaver and Burk (1934) analysis of these results, which was presented elsewhere (Eisenstadt, Grossman, and Klein, 196), showed that the D-isomer acts as a competitive inhibitor of L-aspartate. DISCUSSION These studies on the inhibition of growth by aspartic acid demonstrate that small amounts of D-aspartate can prevent the synthesis of cellular proteins. Similar results illustrating that an amino acid can inhibit growth were reported previously (Rydon, 1948; Meister, 1957), but no mechanisms were postulated. Furthermore, the reuorted inhibitorv effects required quantities of amino acids 1 to 1 times greater than the amounts of aspartate needed in these experiments. The experiments with intact cells indicate that the D-aspartate inhibition of protein synthesis may be a "one-step" inhibition process. Thus, a tenfold increase in D-aspartate concentration causes a tenfold increase in the inhibition of amylase induction. This contention is supported by the experiments wvhich show that L-aspartate can reverse the inhibition either partially or totally, depending on the concentration of L-aspartate used. It was shown here that.1 % casein hydrolysate reverses the effect of.1% D-aspartate by approximately 5%. This level of reversal is probably due to the content, in casein hydrolysate, of L-aspartate, which was reported to be present in concentrations of 6.9 to 7% (Spector, 1956), corresponding to a final concentration of.7% L-aspartate in our experiment;.7% of this amino acid reversed the DL-aspartate effect by 5% (Table 4). Reversal also was elicited by the addition of adenosine to the incubation mixtures, localizing a possible site of D-aspartate inhibition to the synthesis of purines. Adenosine is able to exert a TABLE 6. Utilization of L- and D-aspartate in the formation of AMP by cell-free extracts of Pseudomonas saccharophila Addition* Enzyme Change in Addition* extractt optical density at 28 mps ml L-Aspartate..5.7 No inosine monophosphate No i-aspartate D-Aspartate.5.5 No enzyme...1 * The complete reaction mixture (.8 ml) contained.1 ml of 1 M glycine buffer (ph 8.),.4 ml of.1 M MgCl2,.4 ml of ATP (.1 M),.2 ml of phosphoenol pyruvate (.1 M), approximately 5 units of pyruvate phosphokinase,.1 ml of GTP (.1 M),.6 ml of IMP (.5 M),.7 M of aspartate, and the enzyme preparation. Increase in optical density of the deproteinized solution was determined at 28 m,u after 3 min. t The extract was derived from starch-grown cells, and had a protein content of 5.7 mg/ml. Downloaded from on April 25, 218 by guest

7 VOL. 87, 1964 ASPARTATE INHIBITIONS IN P. SACCHAROPHILA 1361 E (D I.6 z w -J..3- ~~~~~~~IMP ~~~~~~~B FRACTION NUMBER FIG. 4. Nucleotide fractionation on Dowex 1 columns. The reaction mixture was increased ten times over that described in Table 6. At zero time,.6 ml was added to an equal amount of 7% perchloric acid. After 6 min of incubation at 8 C, 4.5 ml were added to an equal amount of 7% perchloric acid. After centrifugation to remove protein, the ph of the supernatant fluids was adjusted to a pink color with phenol red and added to the Dowex (1 by 8; formate form) resin columns, and eluted. Curve A represents the products of the reaction performed with L-aspartate as the amino donor. Curve B represents the products of the reaction with D-aspartate as the amino donor. Curve C represents the reactants before any reaction has taken place. sparing effect on the amount of L-aspartate needed to reverse the effect of a given amount of D-aspartate (Fig. 3). Because the reversals by L-aspartate and adenosine are not additive, the inhibition of protein synthesis by D-aspartate may be the observed result of inhibitions of several different reactions. One reaction may well be the inhibition of purine synthesis (hence, the formation of ATP and other purine nucleoside triphosphates). This, of course, would affect the energy transfer mechanisms in the cell, and the synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA). Another point at which D-aspartate inhibition could occu is at the activation of amino acids prior to trnsfer onto soluble RNA (Loftfield, 1957). Aspartic acid is known to be involved in the syntheses of other amino acids, e.g., threonine, homoserine, and alanine, as well as Krebs cycle intermediates. Introduction of the D-isomer could produce compounds which are not utilizable in such reactions. Addition of D-aspartate to actively growing cultures (Fig. 2) results, after a 2-hr lag, in a change in the rate of protein synthesis from a logarithmic rate to an apparently linear one. Similar results were reported by Cohen and Munier (1959) with cells of Escherichia coli grown in the presence of structural analogues of AMP, IMP B CD A FIG. 5. Electrophoresis of the products of conversion of IMP to adenylosuccinate and AMP by cell-free extracts of Pseudomonas saccharophila. Samples of the experiment described in Fig. 4 were deposited on paper, and electrophoresis was performed with.5 M ammonium formate buffer (ph 8.5) at 8 v for 2 hr. The circled areas indicate regions of ultraviolet absorption. The letters A and B indicate samples from the reaction mixtures of Fig. 4. Downloaded from on April 25, 218 by guest

8 1362 EISENSTADT AND KLEIN J. BACTERIOL. amino acids. These workers concluded that the structural analogues are incorporated into proteins during the linear phase of growth, but bring about the synthesis of proteins which are abnormal by their structure and activity. Although there is no proof of this in the Pseudomonas system, it is possible that some of the amylase or cellular protein, or both, produced in the presence of D-aspartate is inactive enzymatically. Because one possible site of D-aspartate inhibition is in the sy3nthesis of purines or pyrimidines, or both, the change to a linear rate of protein formation may be the result of the immediate inhibition of nucleic acid synthesis. Cells would therefore cease forming new "machinery" for protein synthesis, and the linear rate would be the result of the activity of existing proteinforming sites. The observation that D-aspartate has varying effects on cells that have been grown on different substrates (Table 3) has been tentatively ascribed to the level of aspartate in the free amino acid pool under these conditions. In this connection, amino acid pool levels in growing cultures of Staphylococcus aureus were reported to remain constant during growth (Hancock, 196). That different levels of the free amino acid pool may be responsible for different sensitivities to D- aspartate inhibition is further illustrated by the fact that, in sucrose-grown cells, L-aspartate shows no stimulatory effect on growth as it does in starch-grown cells. Furthermore, a concentration of D-aspartate that will inhibit protein synthesis even in sucrose-grown cells can be reached, although this concentration is tenfold higher than that required to produce a similar effect in starch-grown cells. Finally, extracts of sucrose-grown cells must be dialyzed to lower the background reaction in testing for the formation of AMP, thus indicating larger amounts of reactants available in these preparations. An alternative explanation to account for the observed differences in sensitivity to D-aspartate in cells grown on different substrates is that the D-isomer may be taken up more effectively by some cells than by others. The cell-free experiments indicated that one of the possible reactions inhibited by D-aspartate is the conversion of IMP to AMP. AMP is, of course, a key intermediate in ATP synthesis as well as in the formation of other purine and pyrimidine nucleoside triphosphates. These compounds were shown (Kornberg, 1959) to be intermediates in the biosynthesis of DNA, as well as in the activation of amino acids in protein synthesis (Loftfield, 1957). It is obvious that inhibition of any of these biosynthetic pathways would result in growth inhibition, and eventually in cell death. ACKNOWLEDGMENT This investigation was supported by a grant (G-6442) from the National Science Foundation. LITERATURE CITED BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG A manual of paper chromatography and paper clectrophoresis. Academic Press, Inc., New York. COHEN, G. N., AND R. MUNIER Effets des analogues structuraux d'amino acides sur la croissance, la synthese de proteines, et la synthese d'enzymes. Biochim. Biophys. Acta 31: EISENSTADT, J. M., AND H. P. KLEIN Sulfur incorporation into the a-amylase of Pseudomonas saccharophila. J. Bacteriol. 77: EISENSTADT, J. M., AND H. P. KLEIN Evidence for the de novo synthesis of the alphaamylase of Pseudomonas saccharophila. J. Bacteriol. 82: EISENSTADT, J. M., L. GROSSMAN, AND H. P. KLEIN Inhibition of protein synthesis by D-aspartate and a possible site of its action. Biochim. Biophys. Acta 36: EISENSTADT, J. M., L. GROSSMAN, AND H. P. KLEIN Effects of aspartate on growth and enzyme formation in Pseudomonas saccharophila. Bacteriol. Proc., p HANCOCK, R Accumulation of pool amino acids in Staphylococcus aureus following inhibition of protein synthesis. Biochim. Biophys. Acta 37: HURLBERT, R. B., H. SCHMITZ, A. F. BRUMM, AND V. R. POTTER Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. J. Biol. Chem. 29: KORNBERG, A Enzymatic synthesis of deoxyribonucleic acid. Harvey Lectures Ser. 53, p LANE, E Protein estimation with the Folin- Ciocalteu reagent, p In Methods in enzymology, vol. 3. Academic Press, Inc., New York. LIEBERMAN, I Enzymatic synthesis of adenosine-5'-phosphate from inosine-5'-phosphate. J. Biol. Chem. 223: Downloaded from on April 25, 218 by guest

9 VOL. 87, 1964 ASPARTATE INHIBITIONS IN P. SACCHAROPHILA 1363 LINEWEAVER, H., AND D. BURK The determination of enzyme dissociation constants. J. Am. Chem. Soc LOFTFIELD, R. B The biosynthesis of protein. Progr. Biophys. Biophys. Chem. 8: LUKENS, L. N., AND J. M. BUCHANAN Further intermediates in the biosynthesis of inosinic acid de novo. J. Am. Chem. Soc. 79: MARKOVITZ, A., AND H. P. KLEIN. 1955a. On the sources of carbon for the induced biosynthesis of alpha-amylase in Pmeudomonas 8accharophila. J. Bacteriol. 7.: MAREKOVITZ, A., AND H. P. KLEIN. 1955b. Some aspects of the induced biosynthesis of alphaamylase of Pseudotmonas saccharophila. J. Bacteriol. 7: MARKOVITZ, A., H. P. KLEIN, AND E. H. FISCHER Purification, crystallization, and properties of the a-amylase of Pseudomonas saccharophila. Biochim. Biophys. Acta 19: MEISTER, A Biochemistry of the amino acids. Academic Press, Inc., New York. RYDON, H. N D-Amino acids in microbiological chemistry. Biochem. Soc. Symp. (Cambridge, Engl.) 1AO46. SPECTOR, W. S Handbook of biological data. W. B. Saunders Co., Philadelphia. THAYER, P. S The amylases of Pseudomonas saccharophila. J. Bacteriol. 66: WHELTON, R., AND M. DOUDOROFF Assimilation of glucose and related compounds by growing cultures of Pseudomonas saccharophila. J. Bacteriol. 49: WIAME, J. M., AND M. DOUDOROFF Oxidative assimilation by Pseudomonas saccharophila with C14-labeled substrates. J. Bacteriol. 62: Downloaded from on April 25, 218 by guest