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Biomedical subjects

N Katunuma

Publications and source records attributed to N Katunuma.

At least 235 records · Page 13Linked to original sources
PubMed1979

Effect of a serine protease on isolated myofibrils.

Morphological changes occurred in myofibrils prepared from the glycerinated psoas muscle of rabbit during incubation with a serine protease crystallized from rat skeletal muscle. Two notable phenomena were observed: (1) loss of the Z band in the early stage of incubation and (2) complete disappearance of the A band after swelling of the myofibrils. The results indicate that the serine protease has an action on myofibrils different from that of Ca2+-dependent neutral protease.

Animals↗
PubMed1979

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.

Amino Acids↗
PubMed1979

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.

Amino Acids↗
PubMed1979

Amplification of growth inhibition by glucocorticoid on L5178Y and L1210 lymphoblasts in vivo.

One component of a factor in Proteus mirabilis (Factor 1) which specifically amplifies the induction of several liver enzymes by glucocorticoid in target cells also increases the growth inhibition of glucocorticoid on the ascitic form of L1210 cells and solid tumors of L5178Y lymphoblasts in vivo. The growth of L5U78Y and L1210 lymphoblasts was inhibited by a triamcinolone acetonide dose of over 0.5 to 1.0 mg/kg body weight. Factor 1 increased the inhibitory effect of a triamcinolone acetonide dose of less than 4.0 mg/kg bodyweight but had little effect on the effects of doses of over 4.0 mg. Factor 1 (10 biological units/kg body weight) itself also caused marked inhibition of the growth of these lymphoblasts without affecting the body weight or adrenal gland weight, its effect being equivalent to that of 3- to 4-mg/kg body weight doses of triamcinolone acetonide alone. There was no significant difference in the level of plasma total corticoids or that of plasma adrenocorticotropic hormone between rats treated with 0.9% NaCl solution or those treated with Factor 1, and Factor 1 had no cytotoxic effec on cultured L5178Y lymphoblasts. Thus, Factor 1 may amplify the effect of physiological level of glucocorticoid in mice sufficiently to inhibit the growth of these lymphoblasts without causing any significant side effects to the host animal.

Adrenal Cortex Hormones↗
PubMed1979

Rat liver glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase [EC 2.4.2.14]. Purification and properties.

Glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (amidophosphoribosyltransferase), [EC 2.4.2.14] was purified 1,600-fold from rat liver. The preparation gave two protein bands on acrylamide gel electrophoresis, of which only the main band showed enzyme activity. The molecular weight of the enzyme was estimated to be 215,000, 200,000, and 195,000 by Sephadex G-150 gel filtration, polyacrylamide gel electrophoresis, and sucrose density grandient ultracentrifugation, respectively. The apparent Km values for glutamine and PRPP were 1.24 mM and 0.57 mM, respectively. The concentration-activity curve for PRPP changed from a hyperbolic to a sigmoidal form on addition of AMP or GMP, and this inhibition by AMP was prevented by increasing the PRPP concentration. In the presence of high concentrations of inorganic phosphate, the catalytic activity was decreased and the sensitivity to AMP inhibition was slightly increased. The molecular size of liver amidotransferase was not changed by the addition of PRPP, AMP, or 2-mercaptoethanol. The purified rat liver enzyme has a broad pH-range of activity between 6.5 and 8.5.

Amidophosphoribosyltransferase↗
PubMed1978

Covalent structure of a group-specific protease from rat small intestine. Appendix: crystallographic data for a group specific protease from rat intestine.

"Group-specific" protease (GSP) is a serine protease, obtained from rat small intestine, which preferentially inactivates the apo forms of certain pyridoxal phosphate requiring enzymes. The enzyme contains 224 amino acid residues in a single polypeptide chain and three disulfide bonds. In the present work the covalent structure has been determined and its homologous relationship to those of chymotrypsin, trypsin, and elastase has been established (approximately 33% identity with each). The residues forming the "charge-relay" system of the active site of chymotrypsin (His-57, Asp-102, and Ser-195) are found in corresponding regions in GSP, whereas an alanyl residue at position 176 of GSP corresponds to a residue which participates in the primary substrate binding site in serine proteases (Asp-177 in trypsin; Ser-189 in chymotrypsin). Three disulfide bonds in GSP occur in similar positions in chymotrypsin, trypsin, and elastase. However, GSP lacks a disulfide bond which is present in all known serine proteases (linking Cys-191 to Cys-220 in chymotrypsin). In view of the close proximity of this bond to both the primary and the antiparallel binding sites of various serine proteases, it is likely that its absence in GSP is related to the substrate specificity of this enzyme. It is concluded that GSP diverged from a common ancestor preceding chymotrypsin but following trypsin.

Amino Acid Sequence↗
PubMed1978

A major serine protease in rat skeletal muscle: evidence for its mast cell origin.

The physical, chemical, and immunologic properties of a protease from rat skeletal muscle, proposed to function in the degradation of certain intracellular enzymes, are identical to those of a chymotrypsin-like serine protease isolated from peritoneal mast cells. The results of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 8 M urea indicate that the two rat proteases have identical mobilities corresponding to a molecular weight of 26,000. The relative amino acid compositions of the proteases are nearly identical. Immunodiffusion tests for crossreaction between the muscle protease and antisera directed toward mast cell protease indicate that the former is immunologically identical to mast cell protease. The first 35 amino-terminal residues of the two enzymes are identical and indicate homology of these proteins to other mammalian serine proteases. The sequence analysis of the protease from muscle was extended for an additional 16 positions, and comparison of this amino-terminal sequence with that of a similar enzyme from small intestine showed approximately 75% sequence identity. In contrast, only 40% of the residues in this region of bovine chymotrypsin A were found at corresponding loci in rat muscle protease. It is concluded that the protease from muscle or mast cells is closely related to the enzyme from small intestine which recently was localized in the "atypical" mast cells of gut mucosa [Woodbury, R. G., Gruzenski, G. M. & Lagunoff, D. (1978) Proc. Natl. Acad. Sci. USA 75, 2785-2789].

Amino Acid Sequence↗
PubMed1978

Mode of inhibition of ornithine aminotransferase by L-canaline.

The mechanism of inhibition of ornithine aminotransferase [EC 2.6.1.13] by L-canaline (alpha-amino-gamma-amino-oxybutyric acid) was investigated. Spectral changes of pyridoxal 5'-phosphate in ornithine aminotransferase on addition of L-canaline showed that L-canaline formed an oxime-type compound with pyridoxal 5'-phosphate that had the same spectra as the compound formed on addition of hydroxylamine to the holoenzyme. Kinetic studies indicated that hydroxylamine was a reversible noncompetitive inhibitor, whereas L-canaline was an irreversible inhibitor of ornithine aminotransferase. Other analogs, such as delta-aminovaleric acid and alpha-N-acetyl-L-ornithine, also reacted with the pyridoxal 5'-phosphate of the enzyme, but these compounds were competitive inhibitors with respect to L-ornithine. L-Canaline and hydroxylamine also reacted with pyridoxal 5'-phosphate in pig heart aspartate aminotransferase [EC 2.6.1.1] to produce an oxime, but both of them were reversible and noncompetitive inhibitors of the enzyme. The Ki value of hydroxylamine for ornithine aminotransferase was 4.3 X 10(-7) M and those of L-canaline and hydroxylamine for aspartate aminotransferase were 1.7 X 10(-4) M and 2.2 X 10(-5) M, respectively.

Amino Acids↗
PubMed1978

Serine protease in mice with hereditary muscular dystrophy.

The activities of serine protease, cathepsin B1, ornithine aminotransferase, and aldolase in skeletal muscles of mice with hereditary muscular dystrophy and their normal litter mates were studied. In dystrophic muscle, the specific and total activities of serine protease were much higher than in normal muscle, and the specific activities, but not the total activities, of cathepsin B1 and ornithine aminotransferase were twice those in normal muscle, and several new fragments, which are normally formed by limited proteolysis, were found in dystrophic muscle. When myofibrillar proteins of normal and dystrophic muscles were incubated with highly purified serine protease, their myosin, alpha-actinin and tropomyosin disappeared completely.

Animals↗
PubMed1978

Abnormal expression of a serine protease in human dystrophic muscle.

The activities of serine protease in muscles from normal persons and from patients with progressive muscular and neuromuscular diseases have been determined. A significant increase in the level of serine protease was found in muscle of patients with Duchenne-type muscular dystrophy and with Becker-type muscular dystrophy, but the activity was not increased in muscle of a patient with amyotrophic lateral sclerosis.

Adult↗
PubMed1978

Susceptibilities of various myofibrillar proteins to muscle serine protease.

The ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, alpha-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin greater than tropnin greater than tropomyosin greater than actin. Alpha-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100,000 and 88,000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21,500 and 19,000. Actin was degraded slowly, but no liberated fragment could be detected.

Actinin↗
PubMed1978

The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation.

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.

Animals↗