Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Glutamate dehydrogenase from Bacillus subtilis PCI 219. I. Purification and properties.

Bacillus subtilis PCI 219 has a single glutamate dehydrogenase (GDH) [EC 1.4.1.3] with dual coenzyme specificity [for NAD(H) and NADP(H)]. The enzyme was purified 800-fold from crude extracts of B. subtilis from the post-exponential phase of growth and showed one significant protein band on gel electrophoresis. This band was determined, by activity staining, to have all the GDH nucleotide specificities. Its molecular weight was estimated to be 250,000+/-20,000 by gel filtration, and 270,000+/-30,000 by zone centrifugation in a sucrose density gradient. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that GDH has a subunit size of about 57,000. The pI of GDH was found to bepH 3.7 by isoelectric focusing. GDH exhibited nonlinear kinetics in the reduction of NAD+, and in the reverse direction, the substrate, NH4+, was strongly inhibitory at high concentrations. Purine nucleotides did not affect the activity. The oxidative demination of glutamate was significantly inhibited by the metabolites oxaloacetate and citrate, which acted as allosteric effectors of this enzyme,inhibiting the reaction in one direction. The pH optimum of each of the activities of GDH and the stability of GDH are also reported.     

Purification and properties of Bacillus subtilis inositol dehydrogenase.

Inositol 2-dehydrogenase (EC 1.1.1.18) activity appears during growth of Bacillus subtilis (strain 60015) in nutrient sporulation medium. Its synthesis is induced by myo-inositol and repressed by D-glucose. The enzyme has an apparent molecular weight of 155,000 to 160,000 as determined by sucrose density gradient centrifugation, and it is comprised of four subunits, each having a molecular weight of 39,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme is 4.4 as determined by column isoelectric focusing. The enzyme shows the highest Vmax and lowest Km with myo-inositol as substrate but does not react with scyllo-inositol; it also reacts with the alpha anomer (but not the beta anomer) of D-glucose and with D-xylose. Apparently, the enzyme can remove only the single equatorial hydrogen of the cyclitol or pyranose ring. In contrast to the glucose dehydrogenase of spores, which reacts with D-glucose or 2-deoxy-D-glucose and with NAD or NADP, inositol dehydrogenase requires NAD and does not react with 2-deoxy-D-glucose.     

Purification and properties of Bacillus subtilis aspartate transcarbamylase.

Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.     

Purification and properties of chorismate synthase from Bacillus subtilis.

Chorismatic synthase was purified to apparent homogeneity from Bacillus subtilis. The enzyme required NADPH-dependent flavin reductase, Mg2+, NADPH, and flavin (FMN or FAD) for activity. The molecular weight of chorismate synthase was 24,000 as determined by sodium dedecyl sulfate (SDS)-gel electrophoresis. The enzyme was also isolated in a complex form associated with NADPH-dependent flavin reductase and another enzyme of the aromatic amino acid pathway, dehydroquinate synthase. On SDS-gel electrophoresis, this form was resolved into three bands with molecular weights of 13,000, 17,000, and 24,000. The enzyme complex was easily dissociated and the dissociation resulted in a change in the chromatographic properties of NADPH-dependent flavin reductase which was no longer retained on phosphocellulose whereas chorismate synthase was still adsorbed. Chorismate synthase activity was linear with time and protein concentration, whereas partially purified preparations showed a significant lag period before the reaction took place. Moreover, crude or partially purified enzyme preparations were completely inactivated by dilution and the activity could be recovered by addition of flavin reductase. A possible role of NADPH-dependent flavin reductase in the activation and regulation of chorismate synthase activity is discussed.     

Purification and properties of phytate-specific phosphatase from Bacillus subtilis.

An enzyme which liberates Pi from myo-inositol hexaphosphate (phytic acid) was shown to be present in culture filtrates of Bacillus subtilis. It was purified until it was homogeneous by ultracentrifugation, but it still showed two isozymes on polyacrylamide gel electrophoresis. The enzyme differed from other previously known phytases in its metal requirement and in its specificity for phytate. It had a specific requirement for Ca2+ for its activity. The enzyme hydrolyzed only phytate and had no action on other phosphate esters tested. This B. subtilis phytase is the only known phytate-specific phosphatase. The products of hydrolysis of phytate by this enzyme were Pi and myo-inositol monophosphate. The enzyme showed optimum activity at pH 7.5. It was inhibited by Ba2+, Sr2+, Hg2+, Cd2+, and borate. Its activity was unaffected by urea, diisopropylfluorophosphate, arsenate, fluoride, mercaptoethanol, trypsin, papain, and elastase.     

Purification and properties of mannanase from Bacillus subtilis

Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its M _r was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly>Glx>Ser and Asx>Ala.N.S. Mendoza and L.M. Joson are with Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are with Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is with Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis.

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.     

Purification and properties of a manganese-stimulated endonuclease from Bacillus subtilis.

An endonuclease stimulated by manganese or calcium ions was isolated from Bacillus subtilis. This enzyme attacked double- or single-stranded deoxyribonucleic acid from a variety of sources, including B. subtilis, and was purified from the material released into the medium during protoplast formation. The enzyme appeared as a single peak after glycerol gradient centrifugation and comprised approximately 30 to 35% of the protein in the most purified preparations, as estimated by gel electrophoresis. It had a molecular weight of about 46,000. The mode of action of the enzyme was endonucleolytic, and circular deoxyribonucleic acid was readily cleaved. The enzyme introduced a limited number of both double- and single-strand breaks into native deoxyribonucleic acid, generally yielding products of 1 X 10(6) daltons or more in size. The reasons for this limitation of cleavage were not clear. The activity of the enzyme was inhibited by low levels of Cu2+, Co2+, Hg2+, and Zn2+. It was also inhibited by high concentrations of NaCl. A role for this enzyme in bacterial transormation is suggested.     

Purification and properties of two aromatic aminotransferases in Bacillus subtilis.

Two enzymes which transaminate tyrosine and phenylalanine in Bacillus subtilis were each purified over 200-fold and partially characterized. One of the enzymes, termed histidinol phosphate aminotransferase, is also active with imidazole acetyl phosphate as the amino group recipient. Previous studies have shown that mutants lacking this enzyme require histidine for growth. Mutants in the other enzyme termed aromatic aminotransferase are prototrophs. Neither enzyme is active on any other substrate involved in amino acid synthesis. The two enzymes can be distinguished by a number of criteria. Gel filtration analysis indicate the aromatic and histidinol phosphate aminotransferases have molecular weights of 63,500 and 33,000, respectively. Histidinol phosphate aminotransferase is heat-sensitive, whereas aromatic aminotransferase is relatively heat-stable, particularly in the presence of alpha-ketoglutarate. Both enzymes display typical Michaelis-Menten kinetics in their rates of reaction. The two enzymes have similar pH optima and employ a ping-pong mechanism of action. The Km values for various substrates suggest that histidinol phosphate aminotransferase is the predominant enzyme responsible for the transamaination reactions in the synthesis of tyrosine and phenylalanine. This enzyme has a 4-fold higher affinity for tyrosine and phenylalanine than does the aromatic aminotransferase. Competitive substrate inhibition was observed between tyrosine, phenylalanine, and histidinol phosphate for histidinol phosphate aminotransferase. The significance of the fact that an enzyme of histidine synthesis plays an important role in aromatic amino acid synthesis is discussed.     

A DNA-dependent ATPase from Bacillus subtilis.

A DNA-dependent ATPase (molecular weight 68000) has been purified from extracts of $\text{B. subtilis}$. The enzyme shows specificity for single-stranded DNA and for hydrolysis of ATP (Km 0.4 mM). Similarities with the $\text{rep}$ gene product from $\text{E.coli}$ are discussed.     

Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.