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Biomedical subjects

N Katunuma

Publications and source records attributed to N Katunuma.

At least 199 records · Page 11Linked to original sources
PubMed1983

Homology of amino acid sequences of rat liver cathepsins B and H with that of papain.

The amino acid sequences of rat liver lysosomal thiol endopeptidases, cathepsins B and H, are presented and compared with that of the plant thiol protease papain. The 252-residue sequence of cathepsin B and the 220-residue sequence of cathepsin H were determined largely by automated Edman degradation of their intact polypeptide chains and of the two chains of each enzyme generated by limited proteolysis. Subfragments of the chains were produced by enzymatic digestion and by chemical cleavage of methionyl and tryptophanyl bonds. Comparison of the amino acid sequences of cathepsins B and H with each other and with that of papain demonstrates a striking homology among their primary structures. Sequence identity is extremely high in regions which, according to the three-dimensional structure of papain, constitute the catalytic site. The results not only reveal the first structural features of mammalian thiol endopeptidases but also provide insight into the evolutionary relationships among plant and mammalian thiol proteases.

Amino Acid Sequence↗
PubMed1983

Selective cleavage of peptide bonds by cathepsins L and B from rat liver.

The selective cleavage of peptide bonds by cathepsin L from rat liver was examined with a hexapeptide, luteinizing hormone releasing hormone, neurotensin and oxidized insulin A chain as model substrates. The specificity of cathepsin L was compared with that of cathepsin B. Cathepsin L cleaved peptide bonds that have a hydrophobic amino acid, such as Phe, Leu, Val, and Trp or Tyr, in position P2. A polar amino acid, such as Tyr, Ser, Gly, Glu, Asp, Gln, or Asn, in position P1. enhanced the susceptibility of the peptide bond to cathepsin L, though the importance of the amino acid residue in position P1' was not as great as that of the amino acid in position P2 for the action of cathepsin L. These results suggest that, in contrast to cathepsin B, cathepsin L shows very clear specificity.

Animals↗
PubMed1983

Use of new synthetic substrates for assays of cathepsin L and cathepsin B.

Efficient methods were developed for synthesizing synthetic substrates for assays of cathepsin B and cathepsin L. Several 2-naphthylamide compounds with a blocked NH2-terminus, Suc-Tyr-Met-NA, beta-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met-NA, were specific and sensitive substrates for cathepsin L and cathepsin B; they were not specific for cathepsin L only, because all of them were also hydrolyzed by cathepsin B. Some kinetic constants for the hydrolyses of these three synthetic substrates by cathepsin B and cathepsin L are given.

Animals↗
PubMed1983

Biosynthesis and degradation of gamma-glutamyltranspeptidase of rat kidney.

gamma-Glutamyltranspeptidase (gamma GTP) of rat kidney is an intrinsic glycoprotein bound to the plasma membrane and composed of two nonidentical subunits and an amino-terminal portion of the heavy subunit anchors the enzyme to the membrane. The mechanisms of biosynthesis, post-translational processing and degradation of the enzyme were studied using mono-specific antibody raised to gamma-glutamyltranspeptidase purified from rat kidney. The following results were obtained. Double isotope labeling in vivo showed that gamma-glutamyltranspeptidase is synthesized as a precursor form with a single polypeptide chain of 78,000 daltons, and then processed post-translationally by limited proteolysis, resulting in two subunits of 50,000 and 23,000 daltons. Incorporation of [3H]leucine or [35S]methionine into the precursor form increased until 60 min after their intravenous injection, and a pulse-chase experiment showed that the half life of the precursor form was 53 min. [3H]Fucose and [3H]glucosamine could also be incorporated into the precursor form, showing that glycosylation of the enzyme occurs at the stage of the precursor form. Rat kidney labeled with [3H]fucose was subjected to subcellular fractionation. The Golgi fraction contained the glycosylated precursor form and a small amount of subunits, and the plasma membrane fraction contained mostly subunits with a significant amount of precursor, suggesting that post-translational processing of the precursor occurs on the plasma membrane. The apparent half lives of the native enzyme and the heavy and light subunits were all estimated as 4.3 +/- 0.5 days by labeling with [3H]leucine or [3H]fucose. gamma-Glutamyltranspeptidase has a different turnover rate from aminopeptidase M, which is located in the microvillus membrane close to gamma-glutamyltranspeptidase.

Acyltransferases↗
PubMed1983

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver.

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.

Animals↗
PubMed1983

A new sensitive and convenient assay of ornithine aminotransferase.

A new radioisotopic assay for ornithine aminotransferase with [U-14C]ornithine as a substrate was developed. The dihydroquinazolinium compound formed was extracted from the reaction mixture with n-butanol and an addition of high concentration of sodium sulfate. This assay is sensitive and convenient and more than 50 assays can be done in one day without any special apparatus. The activities of ornithine aminotransferase in rat liver and human lymphoblastoid cells could be measured at nanomole levels by this method. Thus, the assay should be useful for measurement of OAT activity in cultured cells and for studies on the metabolic bases of the different types of ornithine aminotransferase deficiency: vitamin B6-responsive and -nonresponsive types.

Animals↗
PubMed1982

Purification and characterization of thiol proteinase inhibitor from rat liver.

A thiol proteinase inhibitor was purified from rat liver by essentially the same procedure as reported previously (Kominami, E., Wakamatsu, N., and Katunuma, N. (1981) Biochem. Biophys. Res. Commun. 99, 568-575), but without heat treatment. The purified inhibitor appears homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate and displayed no multiple forms. The inhibitor has Mr = 12,500 and contains 50.5% of polar amino acid residues, 9.3% aromatic amino acids, and no tryptophan. The presence of 2 half-cystines/molecule and the absence of free thiol groups indicate that the inhibitor possesses one disulfide bridges. The inhibitor inhibits cathepsin H by forming an enzyme-inhibitor complex in a molar ratio of 1:1. It inhibits most thiol proteinases such as cathepsin H, L, B, and C, papain, and ficin, but not calcium-activated neutral proteinase or serine proteinases or carboxyl proteinases. The inhibitor was found in various rat tissues. Immunological diffusion analysis with anti-liver thiol proteinase inhibitor serum indicated that the rat liver inhibitor is immunologically identical with the inhibitors from other rat tissues. On subcellular fractionation of rat liver, the thiol proteinase inhibitor was recovered in the cytosol fraction.

Amino Acids↗
PubMed1982

Comparison of properties of thiol proteinase inhibitors from rat serum and liver.

Thiol proteinase inhibitors in rat serum were purified and their properties were compared with those of rat liver thiol proteinase inhibitor. The inhibitors in rat serum were separated into three forms (S-1, S-2, and S-3) by linear gradient elution from a DE52 column. One inhibitor (S1) was purified to homogeneity by chromatography on ficin-bound Sepharose and Sephadex G-150 columns. The apparent molecular weights of S1, S2, and S3 on Sephadex G-150 columns were 90,000, 95,000, and 160,000, respectively. Serum thiol proteinase inhibitor and liver thiol proteinase differed in the following: 1) all three forms of serum inhibitor had much higher molecular weights than the liver thiol proteinase inhibitor (Mr = 12,500); 2) no cross-reactivity was observed between serum inhibitors and liver inhibitor in tests with either antiserum inhibitor or anti-liver antiserum; 3) both serum inhibitor and liver inhibitor were specific for thiol proteinases, but had different inhibition spectra; 4) the liver inhibitor did not bind to concanavalin A-Sepharose, whereas the serum inhibitor bound and was eluted with alpha-methyl mannoside. A thiol proteinase inhibitor of high molecular weight detected in tissue homogenates inhibited papain markedly but did not inhibit cathepsin H. Its activity was diminished by perfusion of the organ, indicating that it is derived from serum.

Animals↗
PubMed1982

Modification of rat liver fructose biphosphate aldolase by lysosomal proteinases.

In vivo proteolytic modification of liver aldolase on administration of leupeptin, a thiol proteinase inhibitor of microbial origin, is reported. When leupeptin was injected into rats, the activity of aldolase in the liver decreased to 40% of that in control rats. Molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that a decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. Injection of leupeptin also caused marked increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D and moderate increase of cathepsin B and cathepsin L. Increase in free activity of cathepsin A returned to the level of control rats by 12 hr after injection of leupeptin, whereas 36 hr was required for recovery of decreased aldolase activity. When insulin was coinjected with leupeptin, increase in the activity of free cathepsin A and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to action of a lysosomal protease(s). Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin. The aldolase inactivating proteinase in the lysosomal fraction was inhibited by PMSF and leupeptin and not by pepstatin. Purified cathepsin A (a serine proteinase), cathepsin B and cathepsin L (thiol proteinase) are potent inactivators of aldolase but cathepsin H and cathepsin D are not. Cathepsin A, B and L are involved in inactivation of aldolase in lysosomes. Endogenous thiol proteinase inhibitor which inhibits lysosomal thiol proteinases (cathepsin B, L and H) is found in the cytosol fraction of liver. The level of thiol proteinase inhibitor actually decreased to 60% of that in control rats in leupeptin-treated rats, suggesting that non-thiol proteinase cathepsin A is a major factor in inactivation of aldolase in lysosomes. Not only leupeptin but also other proteinase inhibitors (antipain, E-64-D, chloroquine) caused increase of labilization of the lysosomes and decrease in aldolase activity. Physiological stimuli which are known to induce the labilization of the lysosomal membrane, such as starvation and glucagon, caused slight or no significant increase of activities of free cathepsin A and D and resulted in no apparent change in aldolase activity.

Animals↗
PubMed1982

Immunological studies on cathepsins B and H from rat liver.

The immunological properties of thiol proteinases from rat liver, cathepsin B, H, and L, were examined. Rabbit antisera were prepared against cathepsins B and H from rat liver. Anti-cathepsin B and H ser quantitatively precipitated only the corresponding enzyme protein. On immunodiffusion analysis, anti-cathepsin B serum reacted with cathepsin B, but not with cathepsin H, indicating that the three thiol proteinases are distinct. Immunoelectrophoresis using a mixture of anti-cathepsin B and anti-cathepsin H showed that the precipitin line with cathepsin B moved faster to the anode than with cathepsin H. Immunological diffusion with antisera indicated that rat liver cathepsin B and H were immunologically identical with cathepsins B and H from rat kidney, lung, spleen, brain, and heart, but different from cathepsin B or H from human or rabbit liver.

Animals↗
PubMed1982

Inhibitions of cathepsin B and cathepsin L by E-64 in vivo. II. Incorporation of [3H]E-64 into rat liver lysosomes in vivo.

E-64 is a specific thiol proteinase inhibitor which inhibits lysosomal cathepsins B and L in vitro and in vivo [Hashida, S., Towatari, T., Kominami, E., & Katunuma, N. (1980) J. Biochem. 88, 1805-1811]. This work showed and that E-64 administered in vivo penetrates into lysosomes of the liver, possibly by permeation rather than by endocytosis. When [3H]E-64 was injected into rats i.p., high radioactivity was observed in the serum after a short time and it decreased rapidly. Incorporation of [3H]E-64 into the cytosol fraction of liver also began to decrease 1 h after the injection. Radioactivity in the mitochondrial-lysosomal fraction increased to a maximum after 6 h and then gradually decreased until 72 h. Dose-dependent incorporation of [3H]E-64 into the serum and liver cytosol was observed at all doses tested, but that into the lysosomal fraction increased linearly with doses of only up to 0.l5 mg/100 body weight of E-64. E-64 in the serum and liver cytosol was mostly present in the free form, whereas that in the lysosomal fraction was mostly protein-bound. The time course and dose-response of lysosomal cathepsin B activity to E-64 were closely related to the radioactivity in the protein-bound fraction of the lysosomes. These results suggest that E-64 was transported to the liver cytosol in the free form in the blood and permeated into the lysosomes, where it bound to, and inactivated, E-64 sensitive proteinases.

Animals↗
PubMed1982

Crystallization and properties of human liver ornithine aminotransferase.

Ornithine aminotransferase [EC 2.6.1.13] was purified and crystallized from human liver by a procedure involving heat treatment, chromatographies on DEAE-cellulose, Octyl-Sepharose CL-4B and Sephadex G-200, and crystallization. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated as 44,000 by sodium dodecyl sulfate electrophoresis and as 177,000 by sucrose density gradient centrifugation, indicating that the enzyme is tetrameric. Various properties of the enzyme from human liver are similar to those of the enzyme from rat liver, including its molecular weight, pH optimum, Km values for ornithine, alpha-ketoglutarate and pyridoxal phosphate and specificity for amino acceptor from ornithine. The amino acid compositions of the two enzymes also have certain similarities, but the enzymes differ in electrophoretic mobility and antigenicity: the human enzyme moved more slowly to the anode, and on immunodiffusion analysis, the single precipitin lines formed between anti-human enzyme serum or anti-rat liver enzyme and the enzyme from human liver or lymphoblastoid cells and the rat liver enzyme fused with spur formation.

Amino Acids↗
PubMed1982

Endogenous thiol proteinase inhibitor from rat liver.

A thiol proteinase inhibitor was purified from rat liver by a rapid procedure involving heat treatment of the post-lysosomal fraction, affinity chromatography on papain-Sepharose 4B and Sephadex G-75. The purified inhibitor appeared homogeneous on sodium dodecyl sulfate electrophoresis. The inhibitor had a molecular weight of about 11000 and consists of three forms (pI 4.9, 5.2 and 5.6). The preparation inhibited thiol proteinases but not serine proteinase or aspartate proteinase. Kinetic studies of inhibition of papain by the inhibitor indicate that the inhibition was noncompetitive and pseudo-irreversible. Subcellular fractionations of rat liver indicate that most of all the inhibitor is localized in the cytosol fraction. The physiological role of the inhibitor is discussed.

Amino Acids↗
PubMed1981

Proteolytic modification of rat liver fructose-1,6-bisphosphate aldolase by administration of leupeptin in vivo.

When leupeptin, a thiol protease inhibitor of microbial origin, was injected into rats, the activity of fructose-1,6-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) in the liver decreased to about 60% of that in control rats. However, the concentration of aldolase protein in the liver extracts, measured with a specific antibody obtained with enzyme purified on a phosphocellulose column, remained unchanged. Injection of leupeptin also caused a marked increase in the activities of free lysosomal proteases, such as cathepsin B (EC 3.4.22.1), cathepsin L (EC 3.4.22.-), cathepsin D (EC 3.4.23.5) and lysosomal carboxypeptidase A in the cytosol fraction. A clear inverse relationship between aldolase and cathepsin B activities in the cytosol fraction was demonstrated. The possibility that the less active form of aldolase detected in the livers of leupeptin-treated rats was produced during homogenization was excluded by showing that the aldolase activity was not changed by addition of various protease inhibitors to the homogenization medium., When insulin was coinjected with leupeptin, increase in the activity of free cathepsin L and decrease of activity of aldolase produced by the injection of leupeptin was prevented. These findings indicate that modification of aldolase may be due to the action of a lysosomal protease(s). Enhanced sensitivity of lysosomes to osmotic shock was demonstrated in the livers of leupeptin-treated rats, suggesting that the lysosomal membrane is labilized by administration of leupeptin. Incubation of the purified aldolase with the lysosomal fraction produced the same changes in properties of aldolase as those observed in vivo on injection of leupeptin.

Animals↗
PubMed1981

Properties of fructose-1,6-bisphosphate aldolase inactivating enzymes in rat liver lysosomes.

The intralysosomal localization of the enzymes that catalyse inactivation of rat liver fructose-bisphosphate aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) to a form with antigenic activity was demonstrated. The inactivating enzymes like all other lysosomal markers tested except acid phosphatase, were readily solubilized by hypotonic shock. The inactivating enzyme activity was inhibited by PMSF, TPCK, TLCK and leupeptin, but not by pepstatin. On partial purification of the inactivating activity from the lysosomal fraction by DEAE-Sephadex (A-50) and Sephadex G-100 column chromatographies, it was copurified with lysosomal carboxypeptidase A and cathepsin B (EC 3.4.22.1). Studies on its substrate specificity and sensitivity to inhibitors indicated that cathepsin B and carboxypeptidase A are responsible for almost all the aldolase-inactivating activity in the lysosomal fraction.

Animals↗