Purification of 7-Keto-8-aminopelargonic Acid-7,8-Diaminopelargonic Acid Aminotransferase,an Enzym Involved in Biotin Biosynthesis,from Brevibacterium divaricatum

Vitamin B₆ is synthesized by green Cytisus scoparius callus and green Phellodendron amurense callus cultured on Linsmaier and Skoog Agar-medium with 10^?5m of ±-naphthaleneacetic acid (NAA) and 10^?6 m of 6-benzyladenine (BA). Even when thiamine and inositol were omitted from this medium, the growth and vitamin B₆ content of Cytisus scoparius callus did not change. Vitamin B₆ contents of clones of the calluses varied and were unstable during long-term subculture. Clonal selection was repeated to obtain stable strains with high vitamin B₆ content, and the vitamin B₆ content of one strain of green Cytisus scoparius callus became 4-times higher than that of the green leaves.     

Isolation of New Tobacco Constituents, 8,9-Dihydro-8,9-dihydroxymegastigmatrienone,from Japanese Domestic Suifu Tobacco

The tetradecapeptide of a renin substrate, DRVYIHPFHLLVYS, was used as a substrate for assaying several fungal aspartic and acidic proteinases in the acidic pH range. Aspartic and acidic proteinases froll) Phycomycetes, Mucor and Rhizopus, and Deuteromycotina, Aspergillus and Penicillium, cleaved the tetradecapeptide at its tyrosyl⁴-isoleucyl⁵ (Y⁴-I⁵),histidyI⁶-proly⁷ (H⁶_P⁷) and leucyl¹¹-valyl¹² (L¹¹-V¹²) bonds in the acidic pH range, while acidic proteinases type B and type A-I from Scytalidium lignicolumn, and those from Cladosporium and Basidiomycetes, Pycnoporus sanguineus, and the yeast, Rhodotorula glutinis; showed slightly different specificities towards the tetradecapeptide. Pepsin primarily cleaved the valy³-tyrosyl⁴ (V³-Y⁴) and leucyl¹⁰-leucyl¹¹ (L¹⁰-L¹¹) bonds. All of the aspartic and acidic proteinases of fungal origin tested in the present study have different specificities from that of pepsin.     

Hydrolysis of Proteins by Successive Incubation with Proteinase and Aminopeptidases Mixture

1. A trial test was attempted of complete hydrolysis of peptides and proteins into amino acids by enzymes. “Neutral proteinase” of Bacillus subtilis or “Alkalophilic proteinase” of a Streptomyces sp. was used for preliminary digestion of substrate, and a mixture of three aminopeptidases of Bacillus subtilis was employed for subsequent hydrolysis of proteinase digest.

2. The oxidized insulin B chain was hydrolyzed completely by the method. Several proteins including enzymes which contained no or less cystine and cysteine were also hydrolyzed almost completely.

3. On the other hand, certain glycoproteins were hydrolyzed to leave a few glycopeptides in which all glycomoieties of the proteins were retained. The implications of the results are discussed.     

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.     

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.     

Stage specific requirement of Gfrα1 in the ureteric epithelium during kidney development

Glial cell line-derived neurotrophic factor (GDNF) binds a coreceptor GDNF family receptor α1 (GFRα1) and forms a signaling complex with the receptor tyrosine kinase RET. GDNF-GFRα1-RET signaling activates cellular pathways that are required for normal induction of the ureteric bud (UB) from the Wolffian duct (WD). Failure of UB formation results in bilateral renal agenesis and perinatal lethality. Gfrα1 is expressed in both the epithelial and mesenchymal compartments of the developing kidney while Ret expression is specific to the epithelium. The biological importance of Gfrα1’s wider tissue expression and its role in later kidney development are unclear. We discovered that conditional loss of Gfrα1 in the WD epithelium prior to UB branching is sufficient to cause renal agenesis. This finding indicates that Gfrα1 expressed in the nonepithelial structures cannot compensate for this loss. To determine Gfrα1’s role in branching morphogenesis after UB induction we used an inducible Gfrα1-specific Cre-deletor strain and deleted Gfrα1 from the majority of UB tip cells post UB induction in vivo and in explant kidney cultures. We report that Gfrα1 excision from the epithelia compartment after UB induction caused a modest reduction in branching morphogenesis. The loss of Gfrα1 from UB-tip cells resulted in reduced cell proliferation and decreased activated ERK (pERK). Further, cells without Gfrα1 expression are able to populate the branching UB tips. These findings delineate previously unclear biological roles of Gfrα1 in the urinary tract and demonstrate its cell-type and stage-specific requirements in kidney development.     

Anti‐barnacle Activity of Isocyanides Derived from Amino Acids

Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid‐isocyanides exhibited potent anti‐barnacle activity with EC₅₀ values of 0.07 – 10.34 μg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 μg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid‐isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants.