Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.     

Enzymes of Pyrimidine Metabolism in Mycoplasma mycoides subsp. mycoides

The major pathways of ribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides have been proposed from studies on its use of radioactive purines and pyrimidines. To interpret more fully the observed pattern of pyrimidine usage, cell extracts of this organism have been assayed for several enzymes associated with the salvage synthesis of pyrimidine nucleotides. M. mycoides possessed uracil phosphoribosyltransferase, uridine phosphorylase, uridine (cytidine) kinase, uridine 5'-monophosphate kinase, and cytidine 5'-triphosphate synthetase. No activity for phosphorolysis of cytidine was detected, and no in vitro conditions were found to give measurable deamination of cytidine. Of the two potential pathways for incorporation of uridine, our data suggest that this precursor would largely undergo initial phosphorolysis to uracil and ribose-1-phosphate. Conversely, cytidine is phosphorylated directly to cytidine 5'-monophosphate in its major utilization, although conversion of cytidine to uracil, uridine, and uridine nucleotide has been observed in vivo, at least when uracil is provided in the growth medium. Measurements of intracellular nucleotide contents and their changes on additions of pyrimidine precursors have allowed suggestions as to the operation of regulatory mechanisms on pyrimidine nucleotide biosynthesis in M. mycoides in vivo. With uracil alone or uracil plus uridine as precursors of pyrimidine ribonucleotides, the regulation of uracil phosphoribosyltransferase and cytidine 5'-triphosphate synthetase is probably most important in determining the rate of pyrimidine nucleotide synthesis. When cytidine supplements uracil in the growth medium, control of cytidine kinase activity would also be important in this regard.     

Genetic recombination in Nocardia mediterranei.

The regulation of macromolecular biosynthesis was studied in a temperature-sensitive mutant of Escherichia coli previously identified as containing a single mutation causing a thermolabile sn-glycerol-3-phosphate acyltransferase, the first enzyme of the pathway for phospholipid biosynthesis. When this mutant was shifted to a nonpermissive temperature, phospholipid synthesis, as well as ribonucleic acid, deoxyribonucleic acid, and protein synthesis, decreased in a coordinate manner, suggesting the existence of a common regulatory mechanism. During the same time that the rate of macromolecular synthesis was decreasing at the nonpermissive temperature, the intracellular concentration of adenosine 5'-triphosphate dropped dramatically and the concentration of adenosine monophosphate increased. The concentration of adenosine 5'-diphosphate dropped, but not as markedly. The decrease in macromolecular synthesis and the changes in the adenine nucleotide concentrations can now be attributed to a thermolabile adenylate kinase. The inactivation of adenylate kinase prevented the cell from converting adenosine 5'-monophosphate to adenosine 5'-diphosphate and consequently from making adenosine 5'-triphosphate. This in turn caused a decrease in the rate of macromolecular synthesis and cell growth. Adenylate kinase, therefore, is a key enzyme in controlling the rate of cell growth. The nature of the possible relationship between adenylate kinase and glycerol-3-phosphate acyltransferase is discussed.     

Metabolism of Pyrimidine Nucleotides in a Microorganism: III. Enzymatic Production of Ribose-5-Phosphate from Uridine-5′-Monophosphate by Pseudomonas oleovorans

A study was made to develop a new method for the production of ribose-5-phosphate (R-5-P) from uridine-5'-monophosphate (UMP) by the action of nucleotide-N-ribosidase of Pseudomonas oleovorans, and a suitable medium for the formation of nucleotide-N-ribosidase was established. For the enzymatic conversion of UMP to R-5-P, a cell suspension was employed as the enzyme source. Although degradation of R-5-P, the desired product, occurred during the course of the enzyme reaction, it was prevented by the addition of an appropriate amount of zinc ion and resulted in a stoichiometric conversion of UMP to R-5-P and uracil. Accumulated R-5-P was readily isolated by ion-exchange chromatography of the bacteria-free reaction mixture. Yield of isolated R-5-P was about 60% of the theoretical recovery.     

Indirect inactivation of rat brain cytosolic diacylglycerol kinase by nucleoside-5'-monophosphate

Cytosolic diacylglycerol kinase was inhibited drastically by nucleoside monophosphate. The inhibition was relatively specific for adenosine-5'-monophosphate (5'-AMP), although uridine-5'-monophosphate was also effective. The effect of 5'-AMP on diacylglycerol kinase appeared to be indirect since the degree of inhibition lessened with the dilution of the cytosol and the more purified enzyme failed to respond to 5'-AMP. A 5'-AMP-dependent mediator is proposed to be involved in the inactivation of diacylglycerol kinase.     

9-Beta-D-arabinofuranosyladenine 5'-monophosphate (araAMP) is converted directly to its antivirally active 5'-triphosphate form by 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase.

The antiherpetic agent 9-beta-D-arabinofuranosyladenine (araA) needs to be phosphorylated to its 5'-triphosphate to be effective as an inhibitor of herpes simplex virus replication. Adenosine kinase and deoxycytidine kinase are assumed to convert araA to its 5'-monophosphate. We now found that araAMP is converted to its 5'-triphosphate through a direct pyrophosphate transfer from 5-phosphoribosyl-1-pyrophosphate (PRPP) by PRPP synthetase. The efficiency of phosphorylation of araAMP to araATP is about 5% of that of AMP, as estimated from their Vmax/Km ratios for PRPP synthetase. AraATP is converted to araAMP by PRPP synthetase at a 4-fold higher Km but similar Vmax as ATP.     

Fermentative Accumulation of Guanosine Polyphosphate by Brevibacterium ammoniagenes

Guanosine-3'-diphosphate-5'-monophosphate (3.35 mg/ml), guanosine-3'-diphosphate-5'-diphosphate (MSI) (5.21 mg/ml), and guanosine-3'-diphosphate-5'-triphosphate (MSII) (0.82 mg/ml), in addition to guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate, were accumulated by microbial conversion of 5'-xanthylic acid with a mutant of Brevibacterium ammoniagenes.     

Specific inhibition of phospholipid synthesis in plsA mutants of Escherichia coli.

plsA mutants of Escherichia coli are temperature-sensitive strains which possess two enzymes of abnormal thermolability, sn-glycerol 3-phosphate acyltransferase and adenylate kinase. Phospholipid synthesis is inhibited after shift of plsA mutants to temperatures at the lower end of the nonpermissive temperature range. This inhibition is not due to inactivation of the adenylate kinase activity since nucleic acid (and hence adenosine 5'-triphosphate) synthesis is inhibited only slightly. These results show that in vivo inactivation of the sn-glycerol 3-phosphate acyltransferase can be observed under conditions which allow normal adenylate kinase function.     

Nascent DNA chains synthesized in reversibly permeable cells of mouse thymocytes

Freshly prepared thymocytes continue to synthesize DNA under hypotonic conditions in the presence of 4.5% dextran T-150, the four deoxyribonucleoside triphosphates and ATP. Permeable cells could seal the membrane in a serum-enriched medium within a few hours. 2'-Deoxycytidine 5'-triphosphate is effectively substituted by 5-mercuri-2'-deoxycytidine 5'-triphosphate as a substrate. The newly synthesized mercurated DNA can be separated from cellular DNA and RNA on a thiol-agarose affinity matrix. The rate of incorporation of [3H]thymidine triphosphate into permeable cells is the same as that of the incorporation of [3H]thymidine into intact cells, corresponding to approximately 30% of the rate in vivo. Synthesis in permeable cells reflects DNA replication shown by inhibitors such as 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (aCTP), nalidixic acid and novobiocin and by density shift experiments. More than 80% of the newly synthesized low-molecular-mass DNA, 8-60 nucleotides in length, consists of RNA-linked DNA. This conclusion is based on phosphorylation with [gamma-32]ATP and polynucleotide kinase and rephosphorylation after alkaline hydrolysis. The 5' end of RNA consists of adenylate, guanylate, cytidylate and uridylate residues in a ratio of 4:3:1.5:1.5.     

Pyrimidine metabolism in cotyledons of germinating alaska peas

Cotyledons from Pisum sativum L. cv. Alaska seeds were excised 12, 36, 108, 132, and 156 hours after imbibition in aerated distilled water. They were then incubated under aseptic conditions for 6 hours in solutions containing either uridine-2-¹⁴C or orotic acid-6-¹⁴C. Uridine was more extensively degraded to ¹⁴CO₂ at all germination stages than was orotate, and these rates remained essentially constant at each stage. Incorporation of each compound into RNA increased about 2-fold from the 12th to the 156th hour, although the total RNA present decreased slightly over this interval. Paper chromatography of soluble labeled metabolites produced from orotate showed that the capacity to metabolize this pyrimidine increased markedly as germination progressed. Radioactivity in uridine-5′-P, uridine diphosphate-hexoses, and uridine diphosphate increased most, while smaller or less consistent increases in uridine, uracil, uridine triphosphate, and an unidentified UDPX compound were also observed. The data suggest that orotate metabolism was initially limited by orotidine-5′-phosphate pyrophosphorylase or by 5-phosphoribosyl-1-pyrophosphate. Incorporation of uridine into RNA appeared to be limited at the earliest germination periods by conversion of uridine-5′-P to uridine diphosphate. Thus, during the 1st week of germination the orotic acid pathway and a salvage pathway converting uridine into RNA become activated.     

Interaction of glycogen phosphorylase with 8-azidoadenosine 5'-monophosphate, a photoaffinity analog of AMP

The ability of 8-azidoadenosine 5'-monophosphate (N3AMP) to act as a photoaffinity label for the AMP binding site on glycogen phosphorylase (EC 2.4.1.1) was tested. 8-Azidoadenosine 5'-monophosphate can replace AMP as an allosteric modifier of both phosphorylases a and b; the pH optimum and the extent of activation are comparable to that observed with AMP. 8-Azidoadenosine 5'-monophosphate resembles the natural activator in having a higher affinity for phosphorylase a. The effects of 8-azidoadenosine 5'-monophosphate and AMP on phosphorylase b are additive when each is present at a concentration which gives less than 50% activation. Increasing the concentration of the substrate, glucose 1-phosphate, decreases the apparent activation constant (Ka) for the interaction of 8-azidoadenosine 5'-monophosphate with phosphorylase b. Glucose 6-phosphate is an inhibitor of phosphorylase b with either AMP or 8-azidoadenosine 5'-monophosphate. In the presence of ultraviolet light, 8-azidoadenosine 5'-monophosphate is irreversibly incorporated into phosphorylase a; incorporation at the allosteric site can be reduced if AMP is added prior to irradiation. Under the conditions used in the photolysis experiments, 3--5% of the available AMP sites were labeled with 8-azidoadenosine 5'-monophosphate. The data indicate the potential usefulness of 8-azidoadenosine 5'-monophosphate as a probe for the AMP site on phosphorylase.     

Influence of antimycin a and uncouplers on anaerobic photosynthesis in isolated chloroplasts

Anaerobiosis depresses the light- and bicarbonate-saturated rates of O(2) evolution in intact spinach (Spinacia oleracea) chloroplasts by as much as 3-fold from those observed under aerobic conditions. These lower rates are accelerated 2-fold or more by the addition of 1 mum antimycin A or by low concentrations of the uncouplers 0.3 mm NH(4)Cl or 0.25 mum carbonyl cyanide m-chlorophenylhydrazone. Oxaloacetate and glycerate 3-phosphate reduction rates are also increased by antimycin A or an uncoupler under anaerobic conditions. At intermediate light intensities, the rate accelerations by either antimycin A or uncoupler are inversely proportional to the adenosine 5'-triphosphate demand of the reduction process for the acceptors HCO(3) (-), glycerate 3-phosphate, and oxaloacetate. The acceleration of bicarbonate-supported O(2) evolution may also be produced by adding an adenosine 5'-triphosphate sink (ribose 5-phosphate) to anaerobic chloroplasts. The above results suggest that a proton gradient back pressure resulting from antimycin A-sensitive cyclic electron flow is responsible for the depression of light-saturated photosynthesis under anaerobiosis.     

Biosynthetic Precursors of Some Modified Nucleosides in the Transfer Ribonucleic Acid of Mycoplasma mycoides var. capri

The ribosomal and transfer ribonucleic acid (tRNA) from Mycoplasma mycoides var. capri, grown in a medium containing uridine-((14)C)-5'-triphosphate and cytidine-(5-(3)H)-5'-triphosphate, were isolated and separated. The uridine in both species of RNA was shown to contain (14)C and the cytidine to contain both (3)H and (14)C. Comparison of the labeling of 4-thiouridine and pseudouridine, obtained from an enzymatic digest of the RNA, indicates that their biosynthetic precursor is uridine, not cytidine. It is probable that ribothymidine and dihydrouridine have the same derivation.     

Regulation of the synthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues.

The kinetic and regulatory properties of enzymes involved in the biosynthesis of UDP-D-galactose, UDP-N-acetylglucosamine. GDP-alpha-D-mannose and GDP-beta-L-fucose from D-glucose 6-phosphate in various reticulo-endothelial tissues was studied. The tissues examined include bovine liver, thyroid, spleen, salivary gland, lung, intestine and mesenteric; pulmonary, portal and sub-maxillary lymphnodes. The maximum rates of specific enzymes in these pathways which were slow enough to be rate-limiting in the formation of glycoproteins in these tissues was determined. UDP-D-galactose 4-epimerase was consistently the rate-limiting reaction in the conversion of -d-glucose 6-phosphate to UDP-D-galactose in all of the tissues examined. The series of reactions leading to the formation of GDP-alpha-D-mannose and GDP-beta-L-fucose were limited by the activity of GDP-alpha-D-mannose pyrophosphorylase and GDP-alpha-D-mannose oxidoreductase, respectively. The formation of UDP-N-acetylglucosamine was limited by the rate of the amination reaction which converts -d-fructose 6-hosphate to D-glucosamine 6-phosphate in the presence of glutamine. Several of these rate-limiting enzymes were partially purified from mesenteric lymph node extracts, and their regulatory properties were examined. GDP-alpha-D-mannose was found to be a competitive inhibitor of GDP-alpha-D-mannose pyrophosphorylase. The apparent Km for GTP was 0.06 mM and the Ki for GDP-alpha-D-mannose was 0.03 mM. The concentrations of GTP and GDP-alpha-D-mannose in lymph node extracts were determined to be 0.095 and 0.012 mumol per g, respectively. UDP-N-acetylglucosamine and UDP-D-glucose inhibited D-fructose 6-phosphate amidotransferase in a manner competitive with D-fructose 6-phosphate. The Km for fructose 6-phosphate was 0.3 mM, while the Ki for UDP-D-glucose and UDP-N-acetyglucosamine were determined to be 0.4 mM and 0.045 mM, respectively. The concentrations of these metabolites in lymph node tissue were: UDP-D-glucose, 0.42; UDP-N-acetylglucosamine 0.095; and D-fructose 6-phosphate, 0.073 mumol per g wet weight of tissue. The results obtained in these studies show that specific rate-limiting enzymes in the pathways for the biosynthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues may be subject to cumulative feedback inhibition by the nucleoside diphosphate sugars which are the final products of these systems and the initial precursors of the oligosaccharide units of glycoproteins in these tissues.     

Metabolism of pyrimidine bases and nucleosides in Bacillus subtilis.

In Bacillus subtilis, uracil (Ura), uridine (Urd), and deoxyuridine (dUrd) are metabolized through pathways similar to those of enteric bacteria. Ura is probably converted to uridine 5'-monophosphate by uridine 5'-monophosphate pyrophosphorylase. More than 95% of dUrd added to cultures is converted to Ura and deoxyribose-1-phosphate. Although dUrd kinase activity is detectable in vitro, this enzyme does not seem to play an important role in the metabolism of dUrd. The metabolism of cytosine (Cyt), cytidine (Cyd), and deoxycytidine (dCyd) in B. subtilis appears to be different from that in enteric bacteria. Cytosine cannot be used by Ura-requiring mutants as pyrimidine source. dCyd is deaminated by dCyd-Cyd deaminase or phosphorylated to dCyd nucleotides by dCyd kinase. Cyd is deaminated by dCyd-Cyd deaminase of phosphorylated by Cyd kinase. This Cyd kinase activity has never been reported for B. subtilis.     

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.     

Biosynthesis of cholesteryl glucoside by Mycoplasma gallinarum

The biosynthesis of cholesteryl glucoside by Mycoplasma gallinarum strain J proceeds by the transfer of glucose from uridine-5'-diphosphoglucose to membrane-bound sterol. Galactose also can be coupled to cholesterol via uridine-5'-diphosphogalactose. The reaction is specific for the uridine-5'-diphospho sugars. Enzymatic activity is associated with the membrane. Treatment of the membrane to remove endogenous sterol inactivates the enzyme. Only sterol which has been bound to the membrane participates in the reaction. The optimum pH is about 8.0, and Mg(2+) is required. The reaction is unaffected by nucleotide triphosphate, uridine-5'-monophosphate, and uridine-5'-diphosphate. Reduction of pH to the optimum for beta-glucosidase in the membrane results in loss of synthesized glucoside. The enzyme is saturated at 0.5 mm uridine-5'-diphosphoglucose. The apparent K(m) of 2.05 x 10(-7) indicates a high affinity of the enzyme for the nucleotide sugar.     

Control of CO2 fixation. Regulation of spinach ribulose-5-phosphate kinase by stromal metabolite levels

The effect of stromal metabolites on the light-activated form of ribulose-5-phosphate kinase was studied with the enzyme rapidly extracted from illuminated spinach chlorplasts. In some instances, the effect of metabolites on the dark-inactivated enzyme extracted from darkened chloroplasts was also investigated. (1) The light-activated form of the enzyme is competitively inhibited with respect to ribulose 5-phosphate by 6-phosphogluconate, ribulose 1,5-bisphosphate, 3-phosphoglycerate and phosphate. Also, fructose 1,6-bisphosphate is inhibitory. All these compounds, except ribulose 1,5-bisphosphate, show an increasing inhibitory effect at lower pH values. Therefore, in the presence of these inhibitors, ribulose-5-phosphate kinase becomes strongly pH dependent. These compounds also exert an inhibitory effect on the dark-inactivated enzyme. (2) The assay of stromal levels of 6-phosphogluconate showed that this compound increased dramatically during a light-dark transient. (3) The dark-inactivated form of ribulose-5-phosphate kinase is strongly inhibited by ADP, the inhibition being competitive with respect to ATP. (4) A simulation of stromal metabolite levels in the enzyme activity assay indicates that in illuminated chloroplasts ribulose-5-phosphate kinase attains only about 4% of its maximal activity. When the fully light-activated enzyme is assayed under conditions occurring in the stroma in the dark, the activity is further decreased by a factor of 20. The same assay with the dark-inactivated enzyme yields an activity of virtually zero. (5) These results demonstrate that in the chloroplasts ribulose-5-phosphate kinase can not only be very efficiently switched off in the dark, but also be subjected to fine control during the illuminated state through the action of stromal metabolites.     

Adenylate energy charge in Acholeplasma laidlawii.

Adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate were produced by Acholeplasma laidlawii B-PG9 growing in modified Edward medium. The adenylate energy charge was calculated to be 0.84 +/- 0.07 and ranged from 0.91 to 0.78 during exponential growth (12 to 24 h). During exponential growth, A. laidlawii contained, at 17.5 h, 2.3 X 10(-17) mol of adenosine 5'-triphosphate per colony-forming unit and, at 16 h, 27.3 nmol of adenosine 5'-triphosphate per mg (dry weight). The medium supported a doubling time of 0.95 h. The molar growth yields (Yglucose = grams [dry weight] per mole of glucose used) were 40.2 +/- 3.4 (16 h) and 57.1 +/- 9.7 (20 h) during midexponential growth. A maximum yield of 8.3 X 10(9) colony-forming units was reached at 24 h, when 56% of the initial concentration of glucose had been used. At 40 h, during the stationary phase, 14.95 +/- 3.75 mumol of glucose per ml of medium had been used. At this time, the culture fluids contained 21.86 +/0 mumol of lactate per ml and 3.14 +/- 0.13 mumol of pyruvate per ml.