Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus.

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly.     

The specific modification of histidyl residues of inorganic pyrophosphatase from Bacillus stearothermophilus by photooxidation

Photooxidation of inorganic pyrophosphatase [pyrophosphate phosphohydrolase EC 3.6.1.1] from Bacillus stearothermophilus in the presence of rose bengal resulted in rapid loss of enzymatic activity. The pH profile of the inactivation rate by the photooxidation showed an inflection point around pH 6.8, suggesting the involvement of histidyl residues in the inactivation. Amino acid analysis revealed that the loss of enzymatic activity was accompanied by the destruction of 3 histidyl residues per molecule. The presence of Mg2+ alone afforded partial protection against the inactivation, whereas inorganic pyrophosphate, the substrate, showed almost no protective effect against inactivation. The photooxidation of inorganic pyrophosphatase altered the circular dichroism spectrum and the difference UV spectrum induced by Mg2+ in the near ultraviolet region. These results suggested that histidyl residues appear to be located at the binding site of Mg2+ and may contribute to the conformational change induced by Mg2+.     

The influence of growth temperature on the heat stability of inorganic pyrophosphatase from crude extracts of Bacillus stearothermophilus.

Inorganic pyrophosphatase activity of crude extracts from Bacillus stearothermophilus adapts its thermostability to the growth temperature of the cells. Magnesium ions increase the stability of the enzyme in all cases. Depending on the growth temperature of the cells two pyrophosphatase species differing in their molecular weights and heat stabilities have been detected.     

Two-step purification of tryptophan-accepting tRNA from Bacillus stearothermophilus

Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus. Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [14C]tryptophan per A260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNAtrp were prepared from 750 and 1000 g of frozen cells, respectively. This yield compares favorably with that from other procedures. The pure tRNAtrp has been crystallized under several different conditions.     

The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus.

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.     

beta-Galactosidase from Bacillus stearothermophilus.

Several strains of thermophilic aerobic spore-forming bacilli synthesize beta-galactosidase (EC 3.2.1.23) constitutively. The constitutivity is apparently not the result of a temperature-sensitive repressor. The beta-galactosidase from one strain, investigated in cell-free extracts, has a pH optimum between 6.0 and 6.4 and a very sharp pH dependence on the acid side of its optimum. The optimum temperature for this enzyme is 65 degrees C and the Arrhenius activation energy is about 24 kcal/mol below 47 degrees C and 16 kcal/mol above that temperature. At 55 degrees C the Km is 0.11 M for lactose and 9.8 X 10(-3) M for 9-nitrophenyl-beta-D-galactopyranoside. The enzyme is strongly product-inhibited by galactose (Ki equals 2.5 X 10(-3) M). It is relatively stable at 50 degrees C, losing only half of its activity after 20 days at this temperature. At 60 degrees C more than 60% of the activity is lost in 10 min. However, the enzyme is protected somewhat against thermal inactivation by protein, and in the presence of 4 mg/ml of bovine serum albumin the enzyme is only 18% inactivated in 10 min at 60 degrees C. Its molecular weight, estimated by disc gel electrophoresis, is 215 000.     

Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris

A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2+, and the (Mg-PPi) complex is regarded to be the functional substrate. Free Mg2+ revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2+. Mg2+ is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2+ revealed a non-competitive type of inhibition on the Mg2+-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2+, had to be applied. An enrichment method along with stepwise lowering the concentrations of Triton X-100 and Mg2+ after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2+ was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity.     

Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.     

Phospholipids from Bacillus stearothermophilus

The lipids of Bacillus stearothermophilus strain 2184 were extracted with chloroform-methanol and separated into neutral lipid and three phospholipid fractions by chromatography on silicic acid columns. The phospholipids were identified by specific staining reactions on silicic acid-impregnated paper, by chromatography of alkaline and acid hydrolysis products, and by determination of acyl ester:glycerol:nitrogen:phosphorus molar ratios. The total extractable lipid was 8% of the dry weight of whole cells and consisted of 30 to 40% neutral lipid and 60 to 70% phospholipid. The phospholipid consisted of diphosphatidyl glycerol (23 to 42%), phosphatidyl glycerol (22 to 39%), and phosphatidyl ethanolamine (21 to 32%). The concentrations of diphosphatidyl glycerol and phosphatidyl glycerol were lower in 2-hr cells than in 4- and 8-hr cells. Whole cells were fractionated by sonic treatment and differential centrifugation. The total lipid content, expressed in per cent of dry weight of each fraction was: whole protoplasts, 10%; membrane fraction, 18%; 30,000 x g particulate fraction, 22%; and 105,000 x g particulate fraction, 26%. The relative phospholipid concentrations in each fraction were about the same. As had been previously reported, none of the phospholipid was stable to alkaline hydrolysis.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.     

Overproduction, purification and characterization of GroES and GroEL from thermophilic Bacillus stearothermophilus

Abstract Biofilms containing diverse microflora were developed on bitumen-painted steel and glass tiles suspended in a chemostat model of a water distribution system. Escherichia coli , taken from a naturally occurring biofilm, was transformed with a plasmid containing the anaerobically induced nirB promoter fused to the lacZ reporter gene. The resulting transformant, PRB1, was introduced into the chemostat. After 7 and 13 days, an E. coli strain with an anaerobically induced Lac⁺ phenotype was present in the biofilm. Development of an episcopic differential interference contrast technique combined with UV fluorescence microscopy enabled the simultaneous visualization of E. coli in the biofilm using a fluorescent probe to detect expression of the gusA reporter gene and a lacZ fluorescent probe to monitor anaerobic expression of β-galactosidase from pnirB .     

Cloning, overexpression, purification and crystallisation of ribosomal protein L9 from Bacillus stearothermophilus.

The cloning, sequencing and overexpression of the gene coding for Bacillus stearothermophilus ribosomal protein L9 is described. The sequence corresponds directly to that presented for the protein itself by classical methods, differing at only a few amino acid positions. The purification and crystallisation of the corresponding L9 protein is presented. The crystals are isomorphous to those described for L9 obtained by conventional methods.     

Evolutionary aspects of inorganic pyrophosphatase.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.     

Alkaline inorganic pyrophosphatase from orchid leaves

An alkaline inorganic pyrophosphatase (IP) from leaves of an orchid, Aranda Christine 130 (Arachnis hookerana var. luteola × Vanda Hilo Blue) was purified by acetone precipitation and chromatography on Sephadex G-75 and DEAE-cellulose. The IP gave a single band on non-denaturing gel electrophoresis at pH 8.3 and its M, determined by gel filtration, was 28 000. The pH optimum was 9 and the IP required Mg²⁺ for its activity and stability. The IP exhibited high specificity for PPi and attained a maximum activity at a Mg²⁺: PPi ratio of 10:1. Other cations tested could not replace Mg²⁺ and they were also found to be inhibitory. The IP was also inhibited by EDTA and F^? but not by iodoacetamide.     

Functionally important lysine residues in inorganic pyrophosphatase from E. coli. I. Interaction of inorganic pyrophosphatase with pyridoxal-5'-phosphate

Interaction of inorganic pyrophosphatase from E. coli with pyridoxal-5'-phosphate includes binding of the reagent at the active site through the phosphate group and then a reversible modification of one lysine residue in each of the enzyme's subunit. In the equilibrium state the protein's molecules contain both inactive modified and native subunits. A stable secondary amine is formed upon the sodium borohydride reduction of the modified protein.