Characterization of the receptor binding residues of kisspeptins by positional scanning using peptide photoaffinity probes

Kisspeptins, endogenous peptide ligands for GPR54, play an important role in GnRH secretion. Since in vivo administration of kisspeptins induces increased plasma LH levels, GPR54 agonists hold promise as therapeutic agents for the treatment of hormonal secretion diseases. To facilitate the design of novel potent GPR54 ligands, residues in kisspeptins that involve in the interaction with GPR54 were investigated by kisspeptin-based photoaffinity probes. Herein, we report the design and synthesis of novel kisspeptin-based photoaffinity probes, and the application to crosslinking experiments for GPR54-expressing cells.     

Design and synthesis of biotin- or alkyne-conjugated photoaffinity probes for studying the target molecules of PD 404182

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes equipped with a biotin or alkyne indicator were designed and synthesized based on previous structure–activity relationship studies. These probes retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential proteins were observed to bind PD 404182.     

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.     

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.     

Nitro- and Amino-polysaccharide Formation from Dextran

Dextran was subjected to oxidative scission by periodate, followed by ring closure with nitromethane to form nitrodextran. The nitro group attached to the ring was reduced by LiAlH₄ to yield amino-polysaccharide of which the molecular weight was about 10,000. It became clear that nitrodextran consisted of 3-deoxy-3-nitro-mannopyranoside, -glucopyranoside, -galactopyranoside and -talopyranoside and their molar ratio was 6: 5: 1: 2 as determined by column Chromatographic separation and gas Chromatographic analysis of the methanolyzate of nitro-dextran.     

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’     

Mutation strategies for obtaining chitooligosaccharides with longer chains by transglycosylation reaction of family GH18 chitinase

Enhancing the transglycosylation (TG) activity of glycoside hydrolases does not always result in the production of oligosaccharides with longer chains, because the TG products are often decomposed into shorter oligosaccharides. Here, we investigated the mutation strategies for obtaining chitooligosaccharides with longer chains by means of TG reaction catalyzed by family GH18 chitinase A from Vibrio harveyi (VhChiA). HPLC analysis of the TG products from incubation of chitooligosaccharide substrates, GlcNAc_n, with several mutant VhChiAs suggested that mutant W570G (mutation of Trp570 to Gly) and mutant D392N (mutation of Asp392 to Asn) significantly enhanced TG activity, but the TG products were immediately hydrolyzed into shorter GlcNAc_n. On the other hand, the TG products obtained from mutants D313A and D313N (mutations of Asp313 to Ala and Asn, respectively) were not further hydrolyzed, leading to the accumulation of oligosaccharides with longer chains. The data obtained from the mutant VhChiAs suggested that mutations of Asp313, the middle aspartic acid residue of the DxDxE catalytic motif, to Ala and Asn are most effective for obtaining chitooligosaccharides with longer chains.     

An Arylamidase from Flavobacterium meningosepticum

An arylamidase was purified from Flavobacterium meningosepticum by a series of chromatographies on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-150. The purified enzyme appeared homogeneous on SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be more than 500,000 dalton by using a column of Sepharose 4B and to be 62,000 when checked by SDS-gel electrophoresis. The enzyme was most active at pH 7.5 toward Leu-β-naphthylamide (Leu-β-NA). It catalyzed the hydrolysis of not only various amino acid-β-naphthylamides but also some peptides, but the hydrolysis rate of the latter substrates was quite low. Cys-di-β-naphthylamide was split by this enzyme at an optimal pH of 6.2. Incubation of oxytocin with the enzyme resulted in a decrease in the biological activity, indicating that this arylamidase possesses an oxytocinase (cystyl aminopeptidase)-like activity.