Histidine decarboxylase from rat and rabbit brain: partial purification and characterization.

Histidine decarboxylase, the synthetic enzyme for histamine, was partially purified from regions of rat or rabbit brain rich in the enzyme. The enzyme was purified using ion exchange and hydrophobic column chromatography and chromatofocusing. Approximately 70-fold and 110-fold enrichments were attained from rat and rabbit brain, respectively. Rat and rabbit brain histidine decarboxylase had isoelectric points of pH 5.4 and 5.6, Km values of 80 microM and 120 microM histidine and Vmax values of 210 and 625 pmol histamine formed/hr-mg protein, respectively. The partially purified histidine decarboxylase from both sources was dependent on pyridoxal phosphate for maximal activity and was inhibited by alpha-fluoromethylhistidine, nickel chloride and cobaltous chloride but was not inhibited by impromidine, alpha-methyldopa, DTNB, zinc chloride or mercuric chloride. The enzyme had a broad pH optimum between pH 7.2 and 8.0. These studies provide further information on the characteristics of mammalian histidine decarboxylase from brain.

Variation in the hemolytic activity of Leptospira interrogans serovar canicola.

Leptospira interrogans serovar canicola strain moulton was grown to a high cell density in a protein-free medium with hemin. The hemolysin produced in this culture system showed a greatly expanded spectrum of hemolysis as compared to previous reports of leptospiral hemolysin produced in a more traditional culture system containing serum.

Hemolysin production by Leptospira interrogans serovar canicola in a protein-free medium with hemin.

Leptospira interrogans serovar canicola strain moulton was grown to a high cell density in a protein-free medium. When hemin was added to this medium, hemolysin was produced. Hemolysin was not detected when other porphyrins or cytochrome C were substituted for hemin in the medium.

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli.

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.

Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.

Glycerol protection and purification of Bacillus subtilis glucose dehydrogenase.

Bacillus subtilis glucose dehydrogenase (EC 1.1.1.47) has been purified from sporulating cell extract to apparent homogeneity (as determined by polyacrylamide gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing). The enzyme purified as a single molecular species with no evidence for a multiple form of the enzyme. The B. subtilis glucose dehydrogenase has an apparent isoelectric point of 4.7-4.8 and an apparent Mr = 126,000 and is comprised of four subunits of Mr = 31,500 each. The glucose 2-deoxyglucose and glucosamine substrate specificity of the enzyme is similar to the substrate specificity for B. subtilis spore germination, suggesting that the spore glucose dehydrogenase may play some role in spore germination. The B. subtilis glucose dehydrogenase is extremely dependent on the presence of glycerol or other hydrophobic bond-stabilizing agents (or NAD) for retention of enzymatic activity, and the presence of glycerol (20% w/v) in the extraction and purification buffers was absolutely necessary for the successful purification of this enzyme.

Isolation of a developmental gene of Bacillus subtilis and its expression in Escherichia coli.

Glucose dehydrogenase of Bacillus subtilis is a developmental enzyme that is not found in growing (vegetative) cells but is synthesized after the differentiation process that leads to the production of endospores has started. We have isolated the gene coding for this enzyme from a lambda Charon 4A phage library of B. subtilis DNA. It is transcribed and translated in vegetative cells of the nondifferentiating organism Escherichia coli into enzymatically active glucose dehydrogenase that has the same physicochemical properties as the enzyme produced in B. subtilis during sporulation. Subcloning of the lambda DNA insert into pBR322 plasmid derivatives showed that the glucose dehydrogenase gene was transcribed in E. coli from a promoter within the B. subtilis genome.

Purification and catalytic properties of "thermostable" fumarase from Bacillus stearothermophilus NU-10 and Thermus X-1.

Fumarase (L-malate hydro-lysase E.C.4.2.1.2) was purified from the thermophilic bacteria Bacillus stearothermophilus NU-10 (optimum growth temperature 62-63 degrees C) and Thermus X-1 (optimum growth temperature 70 degrees C). The furmarase from Thermus X-1 is slightly more thermostable and has an "optimum" catalytic reaction temperature of 83 degrees C as compared to 81 degrees C for the B. stearothermophilus enzyme. Increased thermostability of these fumarases permitted an examination of the properties of the enzyme catalyzed reaction at temperatures higher than had previously been possible with the furmarases from mesophilic bacteria or higher plant and animal sources. Beyong the observed thermostability of the thermophilic fumarases, the catalytic properties of thermophilic fumarases were very similar to those observed with bacterial fumarase or the well characterized pig heart fumarase (effect of temperature on substrate affinities, pH optimum, substrate inhibition by fumarate, and Haldane relationship). These similarities suggest that thermophilic enzymes may be useful in the general study of enzyme reaction mechanisms.

Types and distribution of obligate thermophilic bacteria in man-made and natural thermal gradients.

The types and distribution of obligate thermophilic bacteria were found to be similar in a thermal gradient resulting from man-made thermal pollution and the thermal gradients of two natural hot springs located in Colorado.

Purification and properties of threonine deaminase from the X-1 isolate of the genus Thermus.

Threonine deaminase (l-threonine dehydratase EC 4.2.1.16) has been partially purified from a new extreme thermophilic bacterium, Thermus X-1, which is similar to T. aquaticus YT-1. The threonine deaminase of strain X-1 has a maximal rate of reaction at 85 to 90 C and is more thermostable than the threonine deaminase from mesophilic bacteria. The enzyme has an apparent molecular weight of 100,000 to 115,000, a K(m) for l-threonine of 14 mM, a pH optimum of 8.0, and like other threonine deaminases also catalyzes the deamination of serine. However the Thermus X-1 threonine deaminase does not show a strong feedback inhibition by isoleucine. It is suggested that the regulation of the biosynthesis of isoleucine in this extreme theromophile may resemble that reported in Rodospirillum rubrum.

Isolation of a nonpigmented, thermophilic bacterium similar to Thermophilic bacterium similar to Thermus aquaticus.

A thermophilic (70 C), gram-negative bacterium has been isolated that resembles Thermus aquaticus but lacks the type-characteristic yellow carotenoid.

Replacement sporulation of Bacillus subtilis 168 in a chemically defined medium.

A replacement sporulation technique (i.e., the sporulation of vegetative cells upon suspension in an appropriate medium) has been developed for Bacillus subtilis 168 (a transformable Marburg strain of B. subtilis). The replacement sporulation medium used is composed of inorganic salts and 10 mm ammonium lactate or glutamate. The requirement for ammonium lactate or glutamate could also be satisfied by other compounds that are metabolized via the tricarboxylic acid cycle. Sporulation of the suspended vegetative cells was completed by 8 to 10 hr after suspension, and the resulting spores were indistinguishable from spores produced in a conventional growth and sporulation medium. Various physiological changes previously reported to be associated with sporulation (e.g., increase in the level of tricarboxylic acid cycle enzymes and changes in the rates of synthesis of deoxyribonucleic acid, ribonucleic acid, and protein) could also be demonstrated during replacement sporulation.