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J Koyama

Publications and source records attributed to J Koyama.

At least 127 records · Page 7Linked to original sources
PubMed1985

Purification and some properties of a membrane-bound dipeptidyl peptidase IV of guinea pig casein-induced intraperitoneal leukocytes.

Dipeptidyl peptidase IV [EC 3.4.14.5] (DPP-IV) was demonstrated to exist in guinea pig casein-induced, peritoneal polymorphonuclear leukocytes (PMN) as well as contaminating macrophages and lymphocytes through the use of glycyl-L-prolyl-4-methylcoumaryl-7-amide (Gly-Pro-MCA) as a substrate. The DPP-IV of PMN was found to be located both on the plasma membrane and in the cytosol. For characterization, the plasma membrane-bound enzyme isolated from peritoneal cells mainly consisting of PMN was solubilized with 0.1% Triton N101 and purified by DEAE-cellulose chromatography, gel filtration, and affinity chromatographies on concanavalin A-Sepharose 4B and Gly-Pro-Sepharose 4B. The purification was more than 570-fold. The purified enzyme appears to be a noncovalent dimer of the 158K dalton DPP-IV. The pH optimum, isoelectric point and Km value for Gly-Pro-MCA were 7.2, 5.5, and 2.0 X 10(-4)M, respectively. The enzyme was inactivated by diisopropyl fluorophosphate and diprotin A. It was capable of cleaving dipeptidyl-MCA of an X-Pro-MCA type (X: free N-terminal amino acid). These properties are similar to those of DPP-IV purified from other tissues.

Animals↗
PubMed1985

Limited proteolysis of a chemically modified third component of human complement, C3, by cathepsin G of human leukocytes.

We have investigated the limited proteolysis of the third component of complement, C3, by a human leukocyte protease, cathepsin G, by using a chemically modified C3, which was prepared by treatment of C3 with methylamine and a fluorescent thiol reagent, N-(dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and was thus named DACM-C3me. Although native C3 was hardly cleaved by cathepsin G, DACM-C3me was cleaved by cathepsin G into three major fragments, which were termed C3c-G (150,000 daltons, 150 kd), C3d-G (25 kd), and C3a-G (10 kd). C3c-G was composed of four disulfide-linked polypeptide chains of 75 kd, 35 kd, and two 25 kd. C3d-G and C3a-G were single-chain fragments derived from the alpha chain. The N-terminal sequence of C3d-G was determined as Thr-Glu-Asp-Ala-Val-, suggesting that cathepsin G released C3d-G by cleaving a Met-Thr peptide bond which is located at 19 residues toward the N-terminal from the cysteinyl residue forming an internal thiolester linkage in native C3. C3d-G, like C3d-K (a C3d fragment produced by the action of plasma kallikrein), was found to have bioactivities such as leukocytosis-inducing and immunosuppressive activities.

Amino Acid Sequence↗
PubMed1984

Inhibition of the O2- -generating NADPH oxidase of guinea-pig polymorphonuclear leukocytes by rabbit antibody to homologous liver NADPH-cytochrome c (P-450) reductase.

Rabbit antibody highly specific for guinea-pig liver NADPH-cytochrome c (P-450) reductase was found to inhibit dose-dependently the O2- -generating activity of the membrane fraction isolated from phorbol-myristate acetate-stimulated, homologous polymorphonuclear leukocytes. In addition, the antibody also could inhibit the NADPH-cytochrome c (Nitroblue tetrazolium) reductase separated from the membrane fractions and phagosomes of leukocytes by polyacrylamide gel electrophoresis or gel filtration on a Sephacryl S-300 column in the presence of 0.2% Triton X-100. These results demonstrate that the NADPH-cytochrome c reductase in the membrane fractions of leukocytes is antigenically cross-reactive with homologous liver NADPH-cytochrome c reductase, and also suggest that the enzyme of leukocytes participates in the respiratory burst.

Animals↗
PubMed1984

Purification and characterization of a membrane-bound NADPH-cytochrome c reductase capable of catalyzing menadione-dependent O2- formation in guinea pig polymorphonuclear leukocytes.

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2-) in the presence of menadione, was highly purified from membrane fractions of disrupted guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983). Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2-. The purified enzyme preparation contained FAD. When assayed by measuring O2--generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 microM, respectively. The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst.

Animals↗
PubMed1984

Isotype specificity and heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes.

The isotype specificity of Fc-receptors for IgG (Fc gamma-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fc gamma-R monospecific for IgG2, whereas 80% of the cells had two distinct Fc gamma-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fc gamma-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1) rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA (IgG2) rosette-forming T cells bear essentially the Fc gamma-R monospecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fc gamma-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.

Animals↗
PubMed1983

Human C4b-binding protein, C4bp. Chymotryptic cleavage and location of the 48 kDa active fragment within C4bp.

C4bp, a regulator of the classical pathway of complement system, is composed of 6-8 disulfide-linked subunit chains of 75 kDa. Upon incubation with chymotrypsin, C4bp was rapidly cleaved into a nicked C4bp, composed of disulfide-linked 48 kDa and 27 kDa fragments. Subsequent slow cleavage on the 27 kDa fragment resulted in the liberation of the active site-containing 48 kDa fragment from the nicked C4bp. The N-terminal amino acid sequence of the 48 kDa fragment was identical to that of the parent subunit chain of C4bp, indicating that the 48 kDa active fragment was released from the N-terminal side of the parent subunit chain. Based on these results, a possible gross structure of C4bp is proposed.

Amino Acid Sequence↗
PubMed1983

Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes.

Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2- -generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2- and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70 000, accompanying the loss of O2- -generating activity of the vesicles.

Animals↗
PubMed1983

Analysis of proteases involved in phagocytic activity of macrophages through the use of various amino acid esters.

Inhibitory effects of various amino acid esters on the phagocytic activity of guinea pig peritoneal macrophages were studied with sensitized 51Cr-sheep erythrocytes (51Cr-EAb) as well as 125I-alpha-amylase complexed with homologous IgG2 antibody (Ag-Ab complex). The intracellular uptake of 51Cr-EAb was markedly inhibited by N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt) and N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt), but not by N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), N-alpha-acetyl-L-arginine methyl ester (Ac-Arg-OMe), N-alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt) or N-alpha-acetyl-L-lysine methyl ester (Ac-Lys-OMe). When phagocytosis of the Ag-Ab complex was assayed by measuring the amount of digested products released from macrophage cells, Ac-Tyr-OEt also inhibited it as markedly as Ac-Phe-OEt, Ac-Trp-OEt, and Bz-Tyr-OEt did, whereas Bz-Arg-OEt again did not show any effect. The results of analysis of the intracellular fate of the Ag-Ab complex taken up by macrophages through the use of analytical density gradient fractionation of the homogenized cells suggest that Ac-Phe-OEt inhibits the ingestive process since the distribution of Ag-Ab complex showed a single peak, closely accompanying the plasma membrane. Ac-Tyr-OEt, on the other hand, caused a marked accumulation of Ag-Ab complex in the lysosome fraction, reflecting the inhibition of intralysosomal digestion of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1983

Separation of three menadione-dependent, O2--generating, pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes.

Pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes were separated by Sephacryl S-300 gel filtration of the sonicated cells in the presence of 0.2% Triton X-100. Two peaks of NADPH-dependent cytochrome c reductase activities with apparent molecular weights of 400,000 and 120,000 were detected. The replacement of NADPH by NADH, on the other hand, revealed two NADH-dependent cytochrome c reductases with apparent molecular weights of 300,000 and 120,000. The addition of 40 microM menadione to assay mixtures considerably enhanced all the cytochrome c-reducing activities, and the enhancement was accompanied by the formation of superoxide anion (O2-). Analysis of the subcellular localizations of these enzymes by fractional centrifugation demonstrated that the NADPH-dependent enzyme (400,000 daltons) was membrane-bound in nature, and that the NADH-dependent enzyme (300,000 daltons) and the NADPH- and NADH-dependent enzyme (120,000 daltons) existed in the cytosol of leukocytes. Thus, the leukocytes contained at least three types of menadione-dependent, O2--forming enzymes: a membrane-bound NADPH-oxidizing enzyme, and soluble NADH-oxidizing and NAD(P)H-oxidizing enzymes.

Animals↗
PubMed1983

Histidine-rich protein as a possible origin of free amino acids of stratum corneum.

The origin of free amino acids and/or their metabolites of the stratum corneum was investigated by pulse-chase experiments using hairless mice. Two, 24, 48, and 72 hrs after the animals were pulse-chased with 3H-histidine or 3H-arginine, radioactivity was determined in the following three fractions of the epidermis: the 0.1 N HClO4 soluble, ethanol soluble fraction (Fr. I); the 0.1N NClO4 soluble, ethanol insoluble fraction (Fr. II); and the 0.1 N HClO4 insoluble, 8M urea soluble fraction (Fr. III). Radioactivity of the epidermal proteins was also determined in SDS-PAGE gels by the gel slicing method. At first, 3H-histidine and/or 3H-arginine were predominantly incorporated into Fr. III, especially into a certain epidermal protein which showed little mobility on SDS-PAGE. Subsequently, 3H, once incorporated into Fr. III, appeared to shift from Fr. III to Fr. II and, simultaneously, from the top band(s) to a band of 32,000 daltons. As a result of amino acid analysis, a protein of Fr. II was considered a histidine-rich protein corresponding to HRP-II of the new born rats of Ball et al. After 48 hrs, as the radioactivities of Fr. III and Fr. II decreased remarkably, Fr. I gradually increased in radioactivity. The major radioactive substances in Fr. I at 72 hrs were identified as 3H-histidine and 3H-urocanic acid when 3H-histidine was used as a tracer, and as 3H-arginine, 3H-ornithine, and 3H-citrulline in the case of 3H-arginine. The amino acid composition of the histidine-rich protein was very similar to that of the free amino acids of the stratum corneum in the hairless mouse, in which some amino acid metabolites were considered as their precursor amino acids. These results strongly suggest that the free amino acids and/or their metabolites of the stratum corneum might be the final products of a degradation of the histidine-rich protein.

Amino Acids↗
PubMed1982

Limited chymotryptic cleavage of human C4-binding protein: isolation of a carbohydrate-containing core domain and an active fragment.

Human C4 binding protein (C4bp), which is a macromolecular weight (Mr 450,000-590,000) cofactor of C3b/C4b inactivator (I), is composed of 6 or 8 disulfide-linked polypeptide chains of Mr 75,000. Chymotrypsin cleaved C4bp into two major fragments; a large fragment of Mr 160,000, which contained carbohydrate chains and was composed of disulfide-linked polypeptide chains of Mr 25,000, and a small fragment of Mr 48,000, which was a single polypeptide chain and had the cofactor activity of C4bp. These results suggest that chymotrypsin liberates a functional domain-containing Mr 48,000 fragment from each subunit chain of C4bp and yields a core fragment derived from a disulfide-knot domain connecting each subunit chain of C4bp.

Carbohydrates↗
PubMed1982

Effects of protease inhibitors on ingestion and digestion of soluble antigen-antibody complexes by macrophages.

The inhibitory effects of diisopropyl fluorophosphate (DFP) and p-tosyl-L-lysine chloromethyl ketone (TLCK) on the ingestion and digestion of 125I-alpha-amylase [B alpha A, EC 3.2.1.1] complexed with antibody by guinea pig peritoneal macrophages were studied by sucrose density gradient fractionation. When macrophages were exposed to the complex at 37 degrees C and homogenized after washing, most of the trichloroacetic acid (TCA)-insoluble radioactivity was recovered in two fractions which behaved like the plasma membrane and lysosomes in the density gradient, and the TCA-soluble radioactivity was found in the fraction not moving into the gradient. The complex bound to Fc receptors on the cell surface, therefore, was shown to be internalized, transported to the lysosomes, and finally digested to amino acid catabolites. The inhibition with DFP caused an accumulation of the complex in a region between the plasma membrane and lysosomes. This distribution of radioactivity resembled that of cytochalasin B-inhibited macrophages. TLCK also inhibited the digestion of the complex, but the distribution of radioactivity revealed a marked accumulation of the complex in the region of lysosomes. These results provide strong support to the hypothesis that DFP blocks the ingestion of the complex, as in the case of cytochalasin B, whereas TLCK inhibits the intralysosomal proteolysis of the complex.

Amino Acid Chloromethyl Ketones↗
PubMed1982

The immunosuppressive effects of trichothecenes and cyclochlorotine on the antibody responses in guinea pigs.

Immunosuppressive effects of trichothecenes of Fusarium solani and Fusarium nivale, T-2 toxin and fusarenon-X, and also of a mycotoxin of Penicillium islandicum, cyclochlorotine, were studied by measuring the anti-2,4-dinitrophenyl (DNP) antibody responses in guinea pigs immunized with DNP-bovine serum albumin. Among these mycotoxins, T-2 toxin alone suppressed strongly the anti-DNP antibody responses at a certain sublethal dose. With other mycotoxins, no effect was observed at any sublethal doses tested. All of the mycotoxins, on the other hand, inhibited the in vitro blast transformation of guinea pig splenic cells stimulated with lipopolysaccharide of Escherichia coli (B cell mitogen) or concanavalin A (T cell mitogen), when measured by incorporation of 3H-thymidine into DNA. Their inhibitory activities were independent of the sort of mitogen used. As in the case of the antibody responses, T-2 toxin was most potent in reducing the DNA synthesis, and exhibited about 10 and 1000 times as potent inhibitory activity as fusarenon-X and cyclochlorotine, respectively.

2,4-Dinitrophenol↗
PubMed1982

Effects of various protease inhibitors on phagocytosis of antigen-antibody complex by macrophages.

In order to characterize proteases of macrophages involved in phagocytosis, the effects of various protease inhibitors were investigated in measuring the uptake and digestion of Bacillus subtilis alpha-amylase complex of guinea pig IgG2 antibody by homologous peritoneal macrophages. The inhibitory effects were observed with diisopropyl fluorophosphate (DFP), L-1-p-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), 1-chloro-3-tosylamido-7-amino-L-2-heptanone (TLCK), antipain, chymostatin, elastatinal and leupeptins, but not with pepstatins. DFP and TPCK reduced both the amounts of antigen-antibody (Ag-Ab) complex taken up by macrophages and of digested products released from the cells. TLCK, antipain, chymostatin, elastatinal and leupeptins also reduced the amount of digested products, but increased that of Ag-Ab complex associated with macrophages. These results suggest that the inhibitory mechanism of DFP and TPCK toward the digestion of soluble Ag-Ab complex by macrophages differs from those of other inhibitors examined.

Animals↗