Post-proline Dipeptidyl Amino-peptidase from Flavobacterium

Nitrogenase catalyzes not only the reduction of N₂ to NH₃ but also the reduction of C₂H₂ to C₂H₄ and H⁺ ion to H₂ gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C₂H₂ reduction activity strongly but did not inhibit H₂ evolution activity. MA-2, on the contrary, inhibited only H₂ evolution activity. MA-8 inhibited both C₂H₂ reduction and H₂ evolution activity to the same extent.     

Pyrolytic Yields of 2-Amino-9H-pyrido[2,3-b]indole and 3-Amino-1-methyl-5H-pyrido[4,3-b]indole as Matagens from Proteins

An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] from a Klebsiella pneumoniae (Aerobacter aerogenes) mutant produced about 40% maltohexaose (G₆) from short-chain amylose ( =23). Mostly G₆ was produced from maltooligosaccharides larger than G₆ by an exo-mechanism action. It also hydrolyzed G₆ and shorter maltooligosaccharides to give smaller maltooligosaccharides. Its position specificity of action on G₃ through G₈ was studied with maltodextrins specifically labeled at the reducing-end glucose unit with ¹⁴C. The highest frequency of cleavage was at the second bond from the reducing end in G₃ through G₆. For G₇ and G₈, the sixth bond from the nonreducing end of the substrate was cleaved with absolute specificity by the exo-mechanism action.

Kinetic parameters of the exo-maltohexaohydrolase on various substrates were also studied. The Michaelis constant (Km) for short-chain amylose was the smallest among the various substrates examined.

G₆ was also formed from G₄ by a transfer action of the enzyme, with an action pattern dependent on the substrate concentration.     

Inhibitory Effect of Glycine on the Production of Amylase and Proteinase by Bacillus subtilis

It was found that the production of amylase and proteinase by washed cells of Bacillus subtilis var. amyloliquefaciens was inhibited by glycine and its peptides but not by glycine derivatives, in which the free amino group was protected with various groups. Incorporation experiments of glycine-C¹⁴ revealed that about 60 per cent of the radioactivity which had been incorporated into the cells was found in the free amino acid fraction of the bacteria. The inhibitory effect of glycine was easily reversed by the addition of amino acid such as alanine, methionine and glutamic acid. Spermine also caused the reversal of inhibition of the enzyme production by glycine.     

Mutagenicity of the Pyrolysis Products of Ammonium Salts

One molecular species of prothoracicotropic hormone with a molecular weight of about 22, 000 (22K-PTTH) of the silkworm, Bombyx mori, was isolated from 5 × 10⁵ adult heads. The purification procedure consisted of 16 steps including defatting, salt-extraction, fractional precipitations, conventional column chromatographies, and high performance liquid chromatographies. An approximately 5 × 10⁶-fold purification was attained to yield 5.4 μg (0.25 nmol) of the pure hormone with a recovery of 3.3%. Injection of 0.11 ng of the purified 22K-PTTH could elicit adult development in a brainless Bombyx pupa. 22K-PTTH is a basic protein (pI 7.7 ~ 8.7) containing disulfide bond(s). The amino-terminal amino acid sequence of 22K-PTTH was determined to be Gly-Asn-Ile-Gln-Val-Glu-Asn-Gln-Ile-Pro-Asp-Pro-.     

Structural Requirements for Mutagenic Activities of N-Heterocyclic Bases in the Salmonella Test System

The mutagenic activities of quinoline, isoquinoline, phenanthridine, benzo(f)quinoline, benzo(h)quinoline and their α-amino derivatives were compared in relation to the effect of structural changes using the Salmonella typhimurium test system. All mutagenic compounds tested require the liver microsomal fraction for their mutagenic activity. Phenanthridine, two benzoquinolines and quinoline were mutagenic. α-Amination of two benzoquinolines and quinoline resulted to increase their mutagenic activity intensively. Addition of a benzene ring to the benzene moiety of 2-aminoquinoline, so that two carbon atoms are shared, affected distinctly the increase in the mutagenic activity. The co-existence of benzoquinoline series with 2-aminobenzo(f)quinoline showed the clear synergistic action.     

Effect of Emodin on the Mutagenicity of 3-Amino-1-methyl-5H-pyrido[4,3-b]indol toward Salmonella

Mass spectra of N-trifluoroacetyl n-butyl ester (BTFA) of ornithinoalanine (OAL) and lanthionine (LAN) were compared with those of the BTFA derivatives of lysinoalanine (LAL) and the related amino acids. A difference of m/z 14, corresponding to one methylene group, was found in each pair of characteristic fragments between BTFA-OAL and BTFA-LAL. A temperature-programmed gas-liquid chromatography and gas chromatography-mass spectrometry were developed to analyze BTFA-LAN, BTFA-OAL and BTFA-LAL. More LAL and LAN were formed in α-lactalbumin than lysozyme by high-temperature treatment in water. OAL was detected in lysozyme treated at 100° and 120°C in alkali solution, but not in α-lactalbumin.     

Studies on Bacterial Protease

The neutral protease of Bacillus amylosacchariticus was inactivated by low concentrations of several metal-chelating agents and the inactivated enzyme with EDTA restored its activity almost completely by the addition of Zn⁺⁺ or Co⁺⁺ and partially by Fe⁺⁺ or Mn⁺⁺, if these metal ions were added shortly after the EDTA-treatment. The native enzyme was found to contain 0.19% of zinc together with a significant amount of calcium. Parallel increase in specific activity and zinc content of enzyme preparation was observed throughout the purification procedure. The elution pattern of enzyme activity on a CM-cellulose column chromatography also completely coincided with that of protein-bound zinc. A zinc-free inactive enzyme was also reactivated by the addition of zinc or cobalt ions, clearly showing that the neutral protease of B. amylosacchariticus is a zinc mctalloenzyme.     

Studies on Bacteriolytic Enzymes

About 100 soil samples were subjected to screening for microorganisms which were capable of producing lytic enzyme toward Staphylococcus aureus. A strain belonging to Streptomyces was isolated and found to produce lytic enzyme(s) noninduciblly, when grown aerobically at 37°C for 25 hr in a medium containing 7.5% soybean cake extract, 2% dextrin, 0.6% K₂HPO₄, 0.02% each of MgSO₄·7H₂O and KCl, pH 7.0. The crude enzyme preparation was active at pH values of 8.5 and 5.8 toward S. aureus, B. subtilis, L. bulgaricus and Str. faecalis but was completely inert against M. lysodeikticus, indicating the enzyme(s) to be distinguished from other bacteriolytic enzymes of Streptomyces so far reported.     

Studies on Bacterial Protease

Some physicochemical properties and amino acid composition of the alkaline protease of B. amylosacchariticus were determined. The molecular weight and sedimentation coefficient were estimated to be 22,700 and 2.89 s, respectively, and the amino terminal amino acid was identified to be alanine. The enzyme contained 15.9% of nitrogen and was composed of 220 residues of amino acid: lys₆, his₅, arg₃, asp₂₀, thr₁₄, ser₃₇, glu₁₂, pro₁₀, gly₂₅ ala₂₇ val₂₀, met₃, isoleu₁₂, leu₁₂, tyr₉, phe₂, try₃ and amide ammonia₁₆ The results indicate that protein nature and chemical properties of the alkaline protease presented here are distinct from those of alkaline proteases obtained from the other strains of B. subtilis, such as subtilopeptidase A, B and BPN’