Makoto Nishikata

Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules.

Affinity chromatography is very useful in the investigation and characterization of specific interaction between biomolecules. Frontal analysis in affinity chromatography is advantageous from both theoretical and experimental viewpoints. The theory is very simple because we can describe this system by means of a simple equilibrium problem.Chromatographic data can be related easily to the amount of interacting molecules and the equilibrium constant. Useful equations analogous to those of enzyme kinetics can also be derived easily. Thus, frontal affinity chromatography provides information almost identical to that obtainable by enzyme kinetic studies. In addition, this method is more general because it does not depend on enzymatic activity. Experiment is very easy and does not require any special equipment. It is a powerful tool, especially for complicated systems where it has been difficult to find an appropriate method.

Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis

We examined the effect of histatin 5 from human parotid saliva on various proteases. Histatin 5 strongly inhibited a trypsin-like protease produced by Bacteroides gingivalis with an IC50 value of 55 nM. Clostripain was also inhibited (IC50 = 800 nM). Activities of other proteases were not affected significantly. Because B. gingivalis is a suspected periodontal pathogen and its proteolytic enzymes have been considered to be associated with periodontal tissue destruction, it is suggested that salivary histatins play a role as a preventive against periodontal disease.

Characterization of Porphyromonas (bacteroides) gingivalis hemagglutinin as a protease.

A hemagglutinin (HA) was purified to homogeneity from the membrane fraction of the oral bacterium Porphyromonas gingivalis. The HA possessed protease activity hydrolyzing proteins and arginine-containing synthetic substrates. The protease activity was inhibited by thiol-blocking reagents, and hence the HA can be characterized as a cystein protease. The HA functions as an attachment factor and its substrate-binding site is responsible for the attachment to an erythrocyte.

Possibility of Bacteroides gingivalis hemagglutinin possessing protease activity revealed by inhibition studies.

Inhibition of hemagglutinin (HA) activity in a membrane fraction of Bacteroides gingivalis was examined using various compounds. Leupeptin and antipain inhibited the HA activity at nm order. This potency was lost when the aldehyde group of leupeptin was converted to an alcohol moiety. Irreversible protease inhibitors, tosyl‐l‐lysine chloromethyl ketone (TLCK), p‐chloromercuribenzoate (PCMB), and N‐ethylmaleimide (NEM) were also inhibitory. From the inhibition experiments, we speculate that the HA possesses protease activity and that the same site of the molecule participates in the erythrocyte binding and the substrate binding.

A phosphotyrosine-containing quenched fluorogenic peptide as a novel substrate for protein tyrosine phosphatases.

Mca-Gly-Asp-Ala-Glu-Tyr(PO(3)H(2))-Ala- Ala-Lys(DNP)-Arg-NH(2), where Mca is (7-methoxycoumarin-4-yl)acetyl and DNP is 2,4-dinitrophenyl, was synthesized as a fluorogenic substrate for protein tyrosine phosphatases (PTPs). In the peptide, the fluorescent Mca group is quenched efficiently by the DNP group. Although the fluorescence intensity of the substrate was practically unchanged upon PTP-catalysed dephosphorylation, it increased approx. 120-fold upon subsequent treatment with chymotrypsin. Analysis by HPLC showed that chymotrypsin cleaved only the dephosphorylated substrate at the Tyr-Ala bond. Thus with the aid of chymotrypsin, dephosphorylation of the substrate can be measured fluorometrically. A strictly linear correlation was observed between PTP concentration and dephosphorylation rate. The fluorogenic substrate was dephosphorylated by some PTPs much more rapidly than the corresponding (32)P-labelled substrate used for comparison, whereas alkaline phosphatase dephosphorylated the two substrates at similar rates. The fluorogenic substrate is therefore more specific for PTPs than the radiolabelled substrate. The assay with the fluorogenic substrate could be applied to the estimation of kinetc parameters and measurement of PTP activity in crude-enzyme preparations. The lower detection limit of our assay (1 microM substrate in 200 microliter of reaction mixture) was estimated to be 0.2-0.4 pmol, whereas it was estimated to be about 1 pmol in the assay that used (32)P-labelled peptide (specific radioactivity of approx. 1000 c.p.m. /pmol). Our assay is simple, specific, highly sensitive and non-radioisotopic, and hence would contribute greatly to the development of PTP biology.

A sepharose derivative coupled with a leupeptin-like peptide aldehyde, glycylglycyl-l-argininal, and its use as an affinity adsorbent for trypsin

A Sepharose derivative containing a peptide aldehyde, glycylglycyl-L-argininal, the structure of which resembles that of leupeptin was prepared. It was a strong affinity adsorbent for trypsin (EC 3.4.21.4). Bovine trypsin showed higher affinity for this adsorbent at the optimum pH of catalysis (8.2) than at lower pH (5.0). This observation was in good agreement with the pH dependence of the interaction of leupeptin and trypsin (Kuramochi, H., Nakata, H. and Ishii, S. (1979) J. Biochem. 86, 1403-1410). Streptomyces griseus trypsin was also adsorbed while trypsinogen, alpha-chymotrypsin and TLCK-trypsin were not adsorbed. Though anhydrotrypsin, in which Ser-183 is converted to dehydroalanine, was not adsorbed, carbamoylmethylated (His-46) trypsin was adsorbed. Ser-183 proved to be essential for the binding. This adsorbent can also be used as a good tool to study the mechanism of action of leupeptin.

A low calcium environment enhances AP-1 transcription factor-mediated gene expression in the development of osteoblastic MC3T3-E1 cells.

Animals fed a low calcium diet develop hypocalcemia and osteoporotic bone. Earlier we conjectured that a low calcium environment might be one of the factors causing abnormalities in hard tissues. Osteoblastic MC3T3-E1 cells (E1 cells) undergo a process of proliferation and differentiation and then produce small mineralized nodules. In this study, we examined the effects of a low calcium environment on osteoblast-like cells cultured with 10% fetal bovine serum and ascorbic acid. Under the culture condition, nodules with characteristics of normal bone appeared by day 30 regardless of the calcium conditions. However, the low calcium environment enhanced the mRNA expressions of c-fos, c-jun and osteocalcin, a specific marker of the osteoblast phenotype. And the exposure to the low calcium medium inhibited the formation of bone nodules. We further studied the differential expressions of c-fos and c-jun in relation to their responses to serum as a function of phenotypic development in the low calcium environment. Both c-fos and c-jun expressions were highly activated by treatment with epidermal growth factor (EGF), but the magnitude of activation was significantly larger under the low calcium condition than the normal condition at each stage. In addition, DNA-binding activities of activating protein-1 (AP-1), Fos/Jun family dimers, were also accelerated by EGF treatment in the low calcium environment. Our findings suggested that osteocalcin, a bone formation marker, c-fos and c-jun genes, and family protein products (AP-1) interacted to restore the normal cell function which deteriorated in the low calcium environment.

Involvement of prolyl endopeptidase in ascidian fertilization.

Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.

Isolation and characterization of prolyl endopeptidase from eggs of the solitary ascidian Halocynthia roretzi: Comparison with the sperm enzyme

Abstract 1. 1. A prolyl endopeptidase has been highly purified from eggs of the ascidian Halocynthia roretzi by ammonium sulfate fractionation and five chromatographic operations using DEAE-cellulose, DEAE-Sephacel, hydroxylapatite, Sephadex G-150 and Z-Gly-Pro-Leu-Gly-aminohexyl-Sephrose. 2. 2. The molecular weight and isoelectric point of the enzyme were estimated to 66,000 and 5.0, respectively. The pH optimum of the activity was 7.0–7.5. 3. 3. The enzyme was inactivated with diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, Z-Gly-Pro-chloromethyl ketone and sulfhydryl-specific reagents; the susceptibility to these inhibitors was similar to that of the enzyme previously purified from spermatozoa of the same ascidian. 4. 4. The ranking of four prolinal-containing peptides in their inhibitory potencies to the egg enyzme was in good agreement with that to the sperm enzyme.

T-kininogen and a 45 kDa proteinase from Porphyromonas gingivalis

To clarify the pathogenic role of proteinases from Porphyromonas gingivalis, a 45 kDa proteinase was isolated from P. gingivalis culture medium by a combination of gel filtration (Bio-Gel A-0.5 m) and ion-exchange chromatographies (DEAE-Sephacel and SP-Sepharose FF). The enzyme was found to have a molecular mass of 45 kDa by SDS-PAGE and to require mercaptoethanol for its activation. The 45 kDa proteinase cleaved T-kininogen into small fragments, but failed to release kinin. In contrast, T-kininogen inhibited the Arg-amidolytic activity of the 45 kDa proteinase with a Ki of 2 nM. On the other hand, the 45 kDa proteinase did not stimulate the production of PGE2, IL-1beta, and TNF-alpha from the macrophages.