Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Lack of Chemically Induced Mutation in Repair-Deficient Mutants of Yeast

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.     

Inactivation of Aspartic Transcarbamylase in Sporulating Bacillus subtilis: Demonstration of a Requirement for Metabolic Energy

The aspartic transcarbamylase (ATCase) activity of Bacillus subtilis cells disappears rapidly from stationary-phase cells prior to sporulation. ATCase activity does not appear in the culture fluid during the stationary phase; hence the enzyme appears to be inactivated in the cells. The enzyme is inactivated normally in two different mutants lacking proteases; the activity is very stable in crude extracts of cells or in the culture fluid. These results suggest that ATCase is not inactivated by the general proteolysis that occurs in sporulating bacteria. The inactivation of ATCase can be completely inhibited after it has begun by oxygen starvation or addition of fluoroacetate. Inhibitors of oxidative phosphorylation and electron transport also interrupt the inactivation of ATCase. The inactivation of ATCase is very slow in two mutant strains that are deficient in enzymes of tricarboxylic acid cycle. Addition of gluconate to stationary cultures of the mutant strains, which is known to restore depleted adenosine 5'-triphosphate pools in these bacteria, also restores inactivation of ATCase. These experiments support the conclusion that the generation of metabolic energy is necessary for the inactivation of ATCase in stationary cells. ATCase activity is stable in growing cells in which ATCase synthesis is repressed by addition of uracil; the enzyme is inactivated normally, however, when such cells cease growing.     

Studies of Raman Spectra of Water Solutions of Adenosine Tri-, Di-, and Monophosphate and Some Related Compounds

Using a He-Ne CW laser source together with a digital photon counting system, we have obtained well resolved Raman spectra for adenosine mono-, di-, and triphosphate (AMP, ADP, ATP) in aqueous solution. Spectra of these compounds were studied as a function of pH from pH = 0.5 to 13.5 and between 550 and 1700 cm(-1). It was found possible to distinguish spectroscopically between the three phosphates over the pH range studied. A qualitative analysis of vibrational modes responsible for various spectral lines is given. Lines at about 960 and 1100 cm(-1) were found to be good indications of the degree of ionization of the terminal phosphate group.     

Cell Walls and Lysis of Mortierella parvispora Hyphae

Walls of Mortierella parvispora, Pullularia pullulans, Absidia repens, Fusarium oxysporum, and of several Penicillium species varied in their susceptibilities to digestion by glucanase and chitinase. Polysaccharides were present in the residues remaining after enzymatic digestion. Acid hydrolysates of the walls contained glucose, glucosamine, and a small amount of galactose. The walls of M. parvispora, which also contained fucose, were the least digested by these two enzymes. Much of the M. parvispora wall material was resistant to decomposition by a heterogeneous soil community, and viable hyphae were not lysed by a glucanase-chitinase mixture. Walls of this fungus were fractionated, and the chemical composition of the fractions was determined. The chitin which was abundant in one of the fractions was apparently largely shielded from chitinase hydrolysis by a glucan. The ecological significance of these findings is discussed.