Diazepam inhibits potassium-induced aldosterone secretion in adrenal glomerulosa cell

In an attempt to elucidate a possible role of peripheral benzodiazepine receptor in adrenal glomerulosa cell, effect of diazepam on potassium-induced aldosterone secretion was studied using isolated bovine adrenal glomerulosa cell. Diazepam inhibited aldosterone secretion stimulated by 8mM potassium in a dose dependent manner. The ID50 was approximately 14 nM. Although diazepam inhibited potassium action effectively, forskolin-induced aldosterone secretion was not affected by diazepam. These results indicate that peripheral benzodiazepine receptor may have an active role in regulating aldosterone secretion. The voltage dependent calcium channel may be a possible site of benzodiazepine action in this tissue.     

A Monte Carlo sampling method of amino acid sequences adaptable to given main-chain atoms in the proteins

We have developed a computational method of protein design to detect amino acid sequences that are adaptable to given main-chain coordinates of a protein. In this method, the selection of amino acid types employs a Metropolis Monte Carlo method with a scoring function in conjunction with the approximation of free energies computed from 3D structures. To compute the scoring function, a side-chain prediction using another Metropolis Monte Carlo method was performed to select structurally suitable side-chain conformations from a side-chain library. In total, two layers of Monte Carlo procedures were performed, first to select amino acid types (1st layer Monte Carlo) and then to predict side-chain conformations (2nd layers Monte Carlo). We applied this method to sequence design for the entire sequence on the SH3 domain, Protein G, and BPTI. The predicted sequences were similar to those of the wild-type proteins. We compared the results of the predictions with and without the 2nd layer Monte Carlo method. The results revealed that the two-layer Monte Carlo method produced better sequence similarity to the wild-type proteins than the one-layer method. Finally, we applied this method to neuraminidase of influenza virus. The results were consistent with the sequences identified from the isolated viruses.     

Influence of External Osmotic Pressure on Water Permeability and Electrical Conductance of Chara Cell Membrane

Water permeability of the plasma membrane of a Characean internodalcell decreased with an increase in the osmotic pressure of theoutside of the cell, suggesting that the equivalent pore radiusof the water-filled pores becomes smaller with an increase inthe osmotic pressure. In contrast, the apparent membrane resistancedid not increase with an increase in the external osmotic pressure.These facts suggest that ions pass through the membrane mainlyvia pores other than those for bulk water flow. (Received October 22, 1986; Accepted May 22, 1987)     

Metabolisms of Nucleosides in Bacteria

In this study, the authors developed a simplified method for the separation and the quantitative determination of nucleosides and bases, using paper electraophoretic technique. By this method, nucleosides and bases were well separated and determined in a fairly short time. Thus, this method was expected to be as accurate as the published methods and was believed to be a better method for both quantitative determination and detection of the nucleosides and bases in a large number of samples.     

Microbial Formation of D-Pantothenic Acid-α-glucoside

The formation of D-pantothenic acid-α-glucoside (PaA-α-G) was found from D-pantothenic acid (PaA) and maltose in incubation mixtures of microorganisms, especially Saccharomyces yeasts and Sporobolomyces coralliformis IFO 1032. The reaction conditions were investigated for formation of PaA-α-G by resting cells of Spor. coralliformis. The formation of the compound increased with PaA concentration (3~20 mg/ml). The yield was maximum at 5~10 mg/ml of PaA. Cetyl trimethyl ammonium bromide (0.1 %) promoted the formation of PaA-α-G. Sucrose was the optimal α-glucosyl donor. When 30 mg/ml of sucrose was fed to the reaction mixture (initial sucrose, 100 mg/ml; and PaA, 10 mg/ml) at 12-hr intervals, 5.74 mg/ml (3.30 mg/ml as PaA) of PaA-α-G was formed in 48-hr incubation at 28°C with shaking. PaA-α-G was also formed by yeast α-glucosidase, mold maltase and the cell-free extract of Spor. coralliformis. The compound showed approximately 9~10% and 0.1~0.3% (molar ratio) of activity of PaA for Saccharomyces carlsbergensis ATCC 9080 and Lactobacillus plantarum ATCC 8014, respectively. The compound had the same microbiological activity as authentic 4′-O-(α-D-glucopyranosyl)-D-pantothenic acid.     

Studies on Transglycosidation to Vitamin B6 by Microorganisms

Some properties of pyridoxine glucoside-synthesizing enzyme were studied using the partially and highly purified enzyme preparations from Micrococcus sp. No. 431.

The enzyme was stable at pH 7.0 and between 0°C and 30°C. The maximal activity was obtained at pH 8.0 and 37°C. Besides sucrose, phenyl-α-d-glucoside and maltose served as glucosyl donor. Of vitamin B₆ compounds tested, only pyridoxine served as glucosyl acceptor. The enzyme activity was inhibited by PCMB and heavy metal ions, and the inhibition was prevented by 2-mercaptoethanol, indicating the enzyme would be a sulfhydryl enzyme. The activity was not affected by chelating agents and not activated by metal ions.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Studies on the Metabolism of Pantothenic Acid in Microorganisms

The ability of the formation of coenzyme A from pantothenic acid and cysteine in the presence of AMP or ATP was searched in yeasts and bacteria. The result of screening showed that the activity was found in several yeasts and the bacteria belonging to the genera Sarcina, Corynebacterium and Brevibacterium. Particularly, Brevibacterium ammoniagenes IFO 12071 (ATCC 6871) accumulated a large amount of coenzyme A.

Isolation of the reaction products, which were synthesized by Brevibacterium ammoniagenes IFO 12071, were carried out. The isolates were identified as coenzyme A, dephosphocoenzyme A and phosphopantothenic acid.

The possibility for the formation of coenzyme A in a larger amount from pantothenic acid and cysteine was investigated with baker’s yeast under the condition coupled with ATP-generating system.

Effect of various factors affecting the accumulation of coenzyme A was investigated. Among them, glucose concentration and inorganic phosphorus concentration were the most important factors for its accumulation. Coenzyme A was not accumulated without the phosphorylation of AMP to ATP. Several cationic surfactants stimulated the accumulation of coenzyme A.

The amount of coenzyme A accumulated reached about 200 μg per ml of the reaction mixture under the suitable reaction conditions employed.     

Distribution of 7-Keto-8-aminopelargonic Acid Synthetase in Bacteria and the Control Mechanism of the Enzyme Activity

The 7-keto-8-aminopelargonic acid (KAPA) synthetase activities of cell-free extracts from various bacteria were investigated. The experiments on the substrate specificity of KAPA synthetase, using crude cell-free extracts from bacteria having high enzyme activity, showed that l-serine and pyruvic acid could replace l-alanine, but that, when the enzyme was partially purified, these compounds were not effective. Many kinds of amino acids such as l-cysteine, l-serine, d-alanine, glycine, d-histidine, and l-histidine, inhibited the enzyme activity. This inhibition was found to be competitive with l-alanine. Pyridoxal 5′-phosphate, which is a cofactor of the enzyme, also inhibited the enzyme activity at high concentrations. The repression of KAPA synthetase by biotin occurred in Bacillus subtilis and B. sphaericus but not in Micrococcus roseus and Pseudomonas fluorescens, even at a concentration of 1000 mµg per ml of biotin.