Iodinated vancomycin and mucopeptide biosynthesis by cell-free preparations from Micrococcus lysodeikticus

A particulate preparation from Micrococcus lysodeikticus was used to synthesize cell-wall mucopeptide. Radioactive iodinated vancomycin became attached to the preparation simultaneously with a complete inhibition of mucopeptide synthesis. After mucopeptide synthesis had occurred in the absence of antibiotic, the preparation took up more vancomycin, suggesting that new binding sites terminating in acyl-d-alanyl-d-alanine had been produced. The mucopeptide product was divided into a soluble and an insoluble portion, both sensitive to lysozyme. The soluble portion did not combine with vancomycin and hence had presumably lost its terminal d-alanine residues, either by transpeptidation or because of carboxy-peptidase action. The synthesis of both portions was unaffected by the presence of penicillin, but the insoluble part showed increased affinity for vancomycin, thus indicating that penicillin had caused conservation of d-alanyl-d-alanine termini.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

Kinetics of the enzymatic synthesis of benzylpenicillin

The kinetics of the enzymatic synthesis of benzylpenicillin catalysed by penicillin amidase (EC 3.5.1.11) from Escherichia coli have been studied. Both free phenylacetic acid (PAA) and its activated derivative, phenylacetylglycine (PAG), were used in the synthesis as acylating agents for 6-aminopenicillanic acid (6-APA). The catalytic rate constants for synthesis carried out at pH 6.0 were 11.2 and 25.2 s⁻¹, respectively, i.e. they are close and have high absolute values. The main feature of the enzymatic synthesis of benzylpenicillin from phenylacetylglycine, compared with the synthesis from phenylacetic acid, is the shape of the progress curve of antibiotic accumulation. In the former case, benzylpenicillin gradually accumulates until equilibrium is reached. Thus, if the reaction is carried out at the thermodynamically optimum pH of synthesis (low pH), penicillin can be obtained in high yield. In the case of phenylacetylglycine, the kinetic curves are more complex and are characterized by a clear-cut maximum. The presence of the maximum, its value and position on the time axis depend on reagent concentration and on the pH used. A kinetic scheme is proposed which describes well the experimental dependencies. The possibility of using activated acid derivatives in synthesis and the advantages of using computer calculations for process optimization are discussed.     

The interrelationship between mucopeptide and ribitol teichoic acid formation as shown by the effect of inhibitors

1. The biosynthesis of teichoic acid in cell suspensions of two strains of Staphylococcus aureus is partially inhibited by the same low concentrations of penicillin that inhibit mucopeptide synthesis by 90–100%. Further increase in the concentration of the antibiotic by several hundred-fold still fails to cause any greater inhibition of teichoic acid synthesis. 2. Other conditions, such as amino acid deficiency or the presence of cycloserine or 5-fluorouracil, that inhibit mucopeptide synthesis also inhibit teichoic acid formation. 3. The degree of inhibition of teichoic acid synthesis caused by relatively high concentrations (10μg./ml.) of benzylpenicillin depends critically on the age of the culture from which the cell suspensions have been prepared. 4. No significant amounts of soluble teichoic acid have been found in the fluid from cells incubated in the presence of penicillin. 5. A high proportion of the teichoic acid formed in the presence of penicillin can be removed from wall preparations at room temperature by 0·1n-ammonia. This is not true of the teichoic acid formed in the absence of penicillin. 6. The teichoic acid extracted with ammonia from preparations of cell walls made from cells treated with penicillin is excluded from Sephadex G-25, has a low molar ratio of glucosamine to phosphorus and contains muramic acid, alanine, glutamic acid, glycine and lysine. 7. The implications of these results for the mechanism of action of penicillin are discussed.     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

Characteristics of Penicillinase Secretion by Growing Cells and Protoplasts of Bacillus licheniformis

Cultures of the inducible penicillinase-producing strain 749 of Bacillus licheniformis, induced with small amounts of benzylpenicillin, synthesized penicillinase at a high rate for a short period, after which the rate of synthesis slowly declined. During the period of active synthesis, the rate of secretion, as a fraction of the level of cell-bound penicillinase (which is originally high), gradually decreased to a constant level. Chloramphenicol, at a concentration (40 mug/ml) which completely inhibited synthesis of penicillinase, partially inhibited secretion if added during the period of active synthesis. During the phase of reduced synthesis, chloramphenicol was without effect on secretion. Penicillinase secretion, by actively growing cultures of the constitutive penicillinase-producing mutant 749/C, was inhibited by 75% immediately after addition of chloramphenicol. The secretion of part of the penicillinase released during active growth is probably dependent on synthesis of penicillinase, but part of the secreted penicillinase can be released in the absence of synthesis. Protoplasts were obtained from which periplasmic penicillinase has been removed, and these protoplasts were capable of substantial growth and penicillinase synthesis without lysis. At pH 7.5, there was no net incorporation of penicillinase into the cell membrane; the enzyme released was almost entirely of the exo form and was roughly equivalent to the amount of new enzyme formed. At pH 6.0, there was some incorporation of penicillinase into the plasma membrane, and approximately half of the extracellular penicillinase was in the exo form; the remainder perhaps represented membrane fragments. In the presence of chloramphenicol, a small amount of penicillinase was released at pH 7.5 as the exo form; at pH 6.0, practically none was released. We suggest that, with the removal from protoplasts of the periplasmic penicillinase-containing particles, a restriction on secretion has been lifted.     

Dependence of betaine stimulation of vitamin b(12) overproduction on protein synthesis

The betaine-stimulated differential synthesis of vitamin B(12), i.e., the increase in B(12) per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, delta-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B(12) synthesis is a result of its stimulation of synthesis of delta-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B(12) and delta-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

两种抗生素对龙须菜的光合生理效应

探讨了不同浓度的两种抗生素(氯霉素和青霉素G钠)对龙须菜(Gracilaria lemaneiformis)生长、光合作用、呼吸作用、色素含量以及可溶性蛋白含量等生理特性的影响。结果表明:龙须菜的生长受到两种抗生素的影响,但是氯霉素的影响要比青霉素G钠的影响大的多。在氯霉素处理的过程中,光合作用、有效光化学效率(Yield)、藻红藻蓝蛋白以及可溶性蛋白含量都随着氯霉素浓度的升高而显著下降,但是呼吸作用速率由于氯霉素的处理而升高;此外光合色素含量不受氯霉素的影响。在青霉素G钠的处理中,光合作用、有效光化学效率随着青霉素G钠的升高而下降,龙须菜叶绿素a与类胡萝卜素含量随着青霉素G钠浓度的升高而具有升高的趋势,但藻红蛋白、藻蓝蛋白以及可溶性蛋白在各处理组之间均没有表现出一定趋势。这些结果说明,氯霉素对生长的影响主要是光合作用速率的下降,以及有关蛋白合成下降引起,而青霉素G钠对生长的影响可能原因是呼吸作用速率的增加引起。由于龙须菜对氯霉素的敏感性比对青霉素G钠的敏感性更强,氯霉素在基因工程的育种中可能更适合作为选择压力。     

Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus

Plasmid-mediated chloramphenicol resistance in Staphylococcus aureus has been shown to involve acetylation of chloramphenicol by an enzyme induced by growth in the presence of the antibiotic and certain analogues. Analysis of the kinetics of induction has been complicated by (i) the intrinsic inhibitory effects of chloramphenicol on induced enzyme synthesis and (ii) the rapid disappearance of inducer after synthesis of the acetylating enzyme. The compound related to d-threo chloramphenicol which lacks a C(3) hydroxyl substituent (3-deoxychloramphenicol) is a potent inducer of chloramphenicol acetyltransferase but is ineffective as an antibiotic and is not a substrate for the enzyme. The availability of such a "gratuitous" inducer has simplified an analysis of the kinetics of induction of chloramphenicol acetyltransferase. The enzyme from induced bacteria has been purified to homogeneity and has been compared with the analogous enzyme present in E. coli which harbors a resistance transfer factor with the chloramphenicol resistance determinant.     

Action of heliomycin on protein and mucleic acid synthesis in Staphylococcus aureus]

Heliomycin inhibited synthesis of RNA in Staph. aureua which was clearly shown in the study of the antibiotic effect on RNA synthesis in the lag phase of the culture development: heliomycin markedly lowered the maximum RNA level in the biomass observed in the culture at the beginning of the exponential growth. On further growth of the culture heliomycin induced a significant retardness of the process of the natural decrease in the RNA biomass level resulting in increased content of RNA in the cells growing in the presence of heliomycin as compared to the control culture. Retarded natural decrease in the RNA biomass level in the presence of heliomycin was observed also on the antibiotic addition just at the beginning of the exponential growth, during the period of maximum RNA accumulation in the cells. Heliomycin had no effect on synthesis and biomass levels of DNA. Heliomycin inhibited the protein synthesis and was close to chloramphenicol by the level of inhibition of the summation protein synthesis in the biomass. However, comparison of the effect of the above antibiotics on synthesis of beta-galactosidase, an individual enzyme protein showed that heliomycin was much less active as an inhibitor of protein synthesis in comparison to chloramphenicol.     

Inhibition of cell wall synthesis and acylation of the penicillin binding proteins during prolonged exposure of growing Streptococcus pneumoniae to benzylpenicillin

Growing cultures of an autolysis-defective pneumococcal mutant were exposed to [3H]benzylpenicillin at various multiples of the minimal inhibitory concentration and incubated until the growth of the cultures was halted. During the process of growth inhibition, we determined the rates and degree of acylation of the five penicillin-binding proteins (PBPs) and the rates of peptidoglycan incorporation, protein synthesis, and turbidity increase. The time required for the onset of the inhibitory effects of benzylpenicillin was inversely related to the concentration of the antibiotic, and inhibition of peptidoglycan incorporation always preceded inhibition of protein synthesis and growth. When cultures first started to show the onset of growth inhibition, the same characteristic fraction of each PBP was in the acylated form in all cases, irrespective of the antibiotic concentration. Apparently, saturation of one or more PBPs with the antibiotic beyond these threshold levels is needed to bring about interference with normal peptidoglycan production and cellular growth. Although it was not possible to correlate the inhibition of cell wall synthesis or cell growth with the degree of acylation (percentage saturation) of any single PBP, there was a correlation between the amount of peptidoglycan synthesized and the actual amount of PBP 2b that was not acylated. In cultures exposed to benzylpenicillin concentrations greater than eight times the minimal inhibitory concentration, the rates of peptidoglycan incorporation underwent a rapid decline when bacterial growth stopped. However, in cultures exposed to lower concentrations of benzylpenicillin (one to six times the minimal inhibitory concentration) peptidoglycan synthesis continued at constant rate for prolonged periods, after the turbidity had ceased to increase. We conclude that inhibition of bacterial growth does not require a complete inhibition or even a major decline in the rate of peptidoglycan incorporation. Rather, inhibition of growth must be caused by an as yet undefined process that stops cell division when the rate of incorporation of peptidoglycan (or synthesis of protein) falls below a critical value.     

Purification and characterization of the penicillin-binding protein that is the lethal target of penicillin in Bacillus megaterium and Bacillus licheniformis. Protein exchange and complex stability.

The penicillin-binding protein that is thought to be the lethal target of penicillin in Bacillus megaterium (protein 1) has been purified to greater than 95% homogeneity. The membrane-bound penicillin-binding proteins were solubilized with a non-ionic detergent and partially separated from each other by ion-exchange chromatography on DEAE-Sepharose CL-6B. Protein 1 was subsequently purified by covalent affinity chromatography on ampicillin-affinose. Bacillus licheniformis contains an equivalent penicillin-binding protein (protein 1) that can be more readily purified to virtual homogeneity in a one-step procedure. It was separated from the other penicillin-binding proteins by utilizing the observation that in this organism, this particular protein is the only one whose covalent complex with benzylpenicillin subsequently breaks down. Membranes were treated with saturating concentrations of benzylpenicillin followed by the removal of free penicillin and further incubation to allow the complex between benzylpenicillin and protein 1 to break down. The penicillin-binding proteins were then solubilized and applied to a column of ampicillin-affinose to which only protein 1 was bound as the other penicillin-binding proteins still had benzylpenicillin bound to them. Pure protein 1 was eluted from the affinity resin with hydroxylamine. The interaction of benzylpenicillin with purified protein 1 has been studied by separating unbound antibiotic from the benzylpenicillin . protein complex by paper electrophoresis. Benzylpenicillin reacts with the protein rapidly to form a covalent complex and the fully saturated complex has a molar ratio of bound [14C] benzylpenicillin: protein of 0.7:1. The complex breaks down, obeying first-order kinetics, with a half-life of 16 min at 35 degrees C, a value identical to that obtained with the membrane-bound protein. The concentration of benzylpenicillin that results in the formation of 50% of the maximum amount of benzylpenicillin . protein complex is that at which the molar amount of benzylpenicillin present is equal to 50% of the molar amount of penicillin-binding protein, rather than being a measure of any of the kinetic parameters of the binding reaction. This observation may be significant in the interpretation of previous results where the amounts of penicillins needed to kill cells or to inhibit penicillin-sensitive reactions have been expressed as concentrations. The possible importance of the breakdown of beta-lactam . protein complexes in the clinical use of these antibiotics is discussed.     

Transient kinetics of formation of phosphorylated intermediate (EP) by fragmented sarcoplasmic reticulum from bullfrog skeletal muscle: ligand addition sequence-dependent rate of EP formation

In order to obtain a better understanding of Ca2+-activated ATP hydrolysis by sarcoplasmic reticulum, the transient kinetics of phosphorylated intermediate (EP) formation was examined with different sequences of addition of Ca2+ and ATP to GEDTA-added (Ca2+-free) fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. With a short delay line (10-20 ms), the addition sequence of ATP followed by Ca2+ gives rise to a faster EP formation without any lag time. In the reverse sequence of ligand addition, a lag time of 2.5-3 ms was consistently observed irrespective of ATP concentration, and the rate of EP formation was lower. As the preincubation time with Ca2+ became longer, the rate constant for EP formation and the maximum level of EP attainable increased even in the presence of fixed concentrations of Ca2+ and ATP. The rate constant per the unit concentration of EP, however, remained constant, indicating second-order kinetics between Ca2+-activated FSR and ATP. With a preincubation time of 83.6 ms, no lag time was observed. In the addition sequence of ATP and 83.6 ms later Ca2+, a burst in EP formation was observed, which was followed by a usual exponential time-course of EP formation. Similar determinations in the presence of various concentrations of ATP led us to the following estimates of the rates of the reactions leading to EP formation at 15 degrees C. (formula: see text)     

Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium

Hancock, R. (Harvard Medical School, Boston, Mass.), and P. C. Fitz-James. Some differences in the action of penicillin, bacitracin, and vancomycin on Bacillus megaterium. J. Bacteriol. 87:1044-1050. 1964.-Penicillin and cycloserine do not inhibit the growth of protoplasts of Bacillus megaterium, indicating that inhibition of cell-wall synthesis is the only significant process by which they inhibit growth of bacteria. In contrast, bacitracin and vancomycin inhibit growth of protoplasts and bacteria at similar concentrations, indicating that they have important sites of action other than their known inhibition of cell-wall synthesis. At concentrations which inhibit mucopeptide synthesis, penicillin, bacitracin, and vancomycin each cause an increased rate of efflux of K ions from growing bacteria. This effect of penicillin is prevented by chloramphenicol or hypertonic sucrose, whereas the effects of bacitracin and vancomycin are unchanged under these conditions. It is concluded that bacitracin and vancomycin have direct effects on the cytoplasmic membrane, and it is proposed that their inhibition of cell-wall synthesis could be a consequence of these effects. Bacitracin and vancomycin do not compete with penicillin for binding to cells of B. megaterium, a further indication that they have a different primary site of action.     

Cell-wall thickening in Bacillus subtilis. Comparison of thickened and normal walls

1. Incubation of Bacillus subtilis 168 trp in a glucose-amino acids-salts medium lacking tryptophan leads to an inhibition of cellular growth without affecting cell-wall synthesis. The cell walls increased approximately two- to three-fold in thickness and at the same time the amount of mucopeptide in the cells measured chemically increased to about the same extent. 2. Synthesis of mucopeptide and teichoic acid as measured by the extent of incorporation of radioactivity continued linearly for approximately 1h and then stopped. No reason was found for the strictly limited synthesis of the wall polymers. 3. The initial rates of incorporation of [(32)P]P(i) or [(3)H]alanine into teichoic acid and of (3)H-labelled amino acids into mucopeptide were not appreciably inhibited by the addition of chloramphenicol to the glucose-amino acids-salts medium. 4. There was no selective turnover of the mucopeptide synthesized by the cells in a medium lacking tryptophan on resumption of growth in a complete medium. 5. Wall synthesis taking place during the thickening process was similar to normal wall synthesis proceeding in growing cells. Walls of different thicknesses prepared from cells incubated for various times in incomplete medium did not differ qualitatively in composition. The products of autolysis of thickened walls were isolated and the analyses indicated a close similarity in the details of their mucopeptide structure compared with the mucopeptide of cells growing in the exponential phase.     

Dependence of Betaine Stimulation of Vitamin B12 Overproduction on Protein Synthesis

The betaine-stimulated differential synthesis of vitamin B₁₂, i.e., the increase in B₁₂ per increase in dry cell weight, by Pseudomonas denitrificans was inhibited by rifampin and chloramphenicol but not by benzylpenicillin and carbenicillin at concentrations of antibiotic that inhibit growth. The level of the first enzyme of corrin (and porphyrin) biosynthesis, δ-aminolevulinic acid synthetase, was decreased to a much greater degree by rifampin and chloramphenicol than by the penicillins. These data support the concept that betaine stimulation of B₁₂ synthesis is a result of its stimulation of synthesis of δ-aminolevulinic acid synthetase, a labile and presumably rate-limiting enzyme of corrin formation requiring continuous induction. In further support of this hypothesis, it was found that chloramphenicol immediately interfered with both vitamin B₁₂ and δ-aminolevulinic acid synthetase formation, no matter when it was added to the system.     

Reversal by a specific peptide (diacetyl-αγ-l-diaminobutyryl-d-alanine) of vancomycin inhibition in intact bacteria and cell-free preparations

Vancomycin inhibited the growth of Bacillus megaterium, Staphylococcus aureus and Micrococcus lysodeikticus, and in cell-free preparations from B. megaterium it inhibited the formation of mucopeptide and enhanced the accumulation of the lipid intermediate in the biosynthetic pathway. All these inhibitory processes were reversed by the presence of a synthetic peptide analogous to un-cross-linked mucopeptide side chains, namely diacetyl-l-diaminobutyryl-d-alanyl-d-alanine. A considerable amount of vancomycin was found in recovering cells, whether recovery was caused by peptide or took place naturally because a low initial concentration of antibiotic was used. In cell-free preparations pretreated with vancomycin, continued inhibition of mucopeptide synthesis depended on the presence of cell-wall material. This inhibition was also reversible by added peptide.     

Change in the biological properties of salmonellae in acquiring resistance to chloramphenicol]

Mutants of Salmonella typhimurium and Salmonella abony resistant to 40 microgram/ml of chloramphenicol were obtained during selection according to the method of Szybalski on Hottinger broth with increasing concentrations of the antibiotic. By the colony morphology the mutants were divided into 4 groups. The study of the mutant biological properties revealed changes in the growth rate characterized by elongation of the lag-phase and exponential phase, changes in the biochemical activity evident from lower fermentation rate of some carbohydrates and production of hydrogen sulphide and changes in some amino acid dependence. Increased cross resistance to tetracycline and benzylpenicillin and decreased resistance to kanamycin were noted. The LD50 of most mutants was increased as compared to that of the initial strains. Combination of several types of the changes was observed in some mutants. It is supposed that resistance to chloramphenicol in the mutants is due to mutations in several genes. Some of such genes had pleuotropic effect because of the changes in the structure of the ribosome 50S subunits.     

Stopped flow and steady state kinetic studies of the effects of metabolites on the soluble form of NADP+:isocitrate dehydrogenase

The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. Stopped flow kinetics showed a distinct lag time, followed by attainment of an apparently linear final velocity. Direct nonlinear regression analyses of the reaction progress curves allowed for the calculation of the rate constant (kappa) for the transition of the enzyme from an inactive to an active form; this transition is best catalyzed by its metal-substrate complex. Preincubation with metal-substrate or metal-citrate nearly abolished the lag by increasing kappa 10-fold. In steady state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both activate and inhibit the enzyme. This analysis suggested: (a) activation by binding to sites with an average dissociation constant of 0.25 mM; (b) inhibition by binding to sites with an average dissociation constant of 3.83 mM; and (c) modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+:isocitrate dehydrogenase.