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Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 217 records · Page 12Linked to original sources
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

Purification and properties of proteinase B from yeast.

Proteinase B (EC 3.4.22.9) was purified from commercial baker's yeast and from wild type strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis. For large scale purification a procedure was developed involving hydrophobic chromatography on octyl-Sepharose 4B and gel filtration on Sephadex G-100. A rapid purification of small amounts of proteinase B was achieved by affinity chromatography on the nitrated proteinase B inhibitor, immobilized on CH-Sepharose according to Bünning and Holzer (Bünning, P. and Holzer, H. (1977). J. Biol. Chem. 252, 5316-5323). The enzyme prepared from all three sources appeared to be homogeneous and exhibited a molecular weight of 33 000 in SDS-polyacrylamide gel electrophoresis. Homogeneity and molecular weight were confirmed for the enzyme from baker's yeast by ultracentrifugation studies. Polyacrylamide gel electrophoresis without SDS and electrofocusing however, indicated microheterogeneity of the proteinase B activity. The aminoterminal residue of the enzyme was found to be glycine. Proteinase B turned out to be a glycoprotein, containing 8-9% neutral sugars and 1.5% amino sugars. The enzyme is blocked by p-hydroxymercuribenzoate and by the serine proteinase inhibitors DFP and PMSF. Among the proteinase inhibitors from microbial origin, chymostatin and antipain were the most powerful inhibitors of proteinase B.

Amino Acids↗
PubMed1981

The substrate specificity of proteinase B from baker's yeast.

The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The Km value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only 1/20 that of chymotrypsin A. Km and kcat for Bz-Arg-OEt are 1/50 and 1/7 as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.

Binding Sites↗
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↗
PubMed1981

Rat liver thiol proteinases: cathepsin B, cathepsin H and cathepsin L.

Data on following points of lysosomal thiol proteinases (cathepsins B, H and L) from rat liver are described in this paper: Partial amino acid sequence of cathepsin B, substrate specificity of cathepsin L, immunological studies of cathepsin B and H and effectiveness of E-64, specific thiol proteinase inhibitor in vivo.

Amino Acid Sequence↗
PubMed1980

Inhibitions by E-64 derivatives of rat liver cathepsin B and cathepsin L in vitro and in vivo.

The mechanism of inhibition of cathepsin B [EC 3.4.22.1] and cathepsin L [EC 3.4.22.-] by E-64 was investigated. Kinetic studies indicated that E-64 was an irreversible inhibitor of these enzymes. [3H]E-64 is incorporated into cathepsin B in a one/one molar ratio in parallel with inactivation of the enzyme. Titration of one of the 10 SH groups of native cathepsin B with 2,2'-dithiodipyridine resulted in complete loss of enzyme activity. Decrease of titratable SH groups and activity of cathepsin B was proportional to the concentration of E-64 added, indicating that E-64 binds to an equimolar amount of active SH residues of cathepsin B. The effects of E-64 and its derivatives on lysosomal cathepsin B and cathepsin L in rat liver were studied in vitro and in vivo. The D form of E-64 inhibited the cathepsin both in vitro and in vivo, although its inhibitory effects were less than those of E-64-(L). E-64-b(RR), in which the terminal agmatine of E-64 is replaced by leucine, was as active as E-64-(L) in vitro, but was completely inactive in vivo. Among the E-64 derivatives tested, E-64-c(SS), in which the terminal agmatine of E-64 is replaced by isoarylamide, showed strong inhibitory activity in vivo, like E-64-(L).

Agmatine↗
PubMed1978

Modes of proteolysis of cystathionase and ornithine aminotransferase by serine protease from the rat small intestine.

The mechanisms of proteolysis of two apo-forms of pyridoxal enzymes, cystathionase [EC 4.2.1.15] and ornithine aminotransferase [EC 2.6.1.13] by serine protease from the rat small intestine were compared. The apo-forms of these two pyridoxal enzymes are susceptible to the serine protease, whereas the holo-forms of the enzymes are not. Pyridoxal phosphate, the coenzyme of these two enzymes, prevented their inactivation by the serine protease. The difference in susceptibility of the apo- and holo-forms to the serine protease was due to the difference in their conformations. The time course of inactivation of cystathionase by the protease was apparently biphasic. During the first phase of inactivation, disappearance of the band corresponding to the native protomer, with a molecular weight of 47,000, was accompanied by accumulation of new material with a molecular weight of 39,000. In the late stage of proteolysis, extensive degradation of the large molecular weight intermediate was observed. This large intermediate product was isolated and found to compete with intact cystathionase as a substrate for the protease. Proteolysis of cystathionase was accompanied by both dissociation of the enzyme molecule and loss of its antigenicity. Limited proteolysis of ornithine aminotransferase apoenzyme by the serine protease resulted in formation of a large molecular weight product similar to the native apoenzyme, but with nicks in the molecule. Dodecylsulfate-polyacrylamide gel electrophoresis showed that the apoenzyme is degraded to intermediate forms (molecular weight 41,500 and 15,000) and later, to stable forms (molecular weight 25,500 and 13,500).

Animals↗
PubMed1977

A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 1. Purification, properties and biological action.

1. A factor, which amplifies the inductions of several liver enzymes by glucocorticoid, was partially purified from Proteus mirabilis from rat intestine. The factor (amplifier) was completely inactivated by alpha-glucosidase, but not by other glycoside hydrolases, proteases, nucleases or phosphatases tested; it was also hydrolysed by HCl with liberation of reducing sugars. Thus the oligosaccharide in this factor seems to be essential for the amplification. 2. In adrenalectomized rats the amplifier increased the inductions of several liver enzymes, such as tyrosine aminotransferase and leucine aminotransferase, by glucocorticoid. But it did not amplify the induction of tyrosine aminotransferase by glucagon or insulin or the activities of enzymes that are not induced by glucocorticoid. The amplifier by itself did not have any glucocorticoid-like action in adrenalectomized rat. These results show that the amplifier specifically increases the inductions of liver enzymes by glucocorticoid. 3. Since similar amplification was also observed in isolated perfused liver and cultured hepatoma cells in vitro, the amplifier seems to act directly on the target organ or cells.

Animals↗
PubMed1976

Studies of new intracellular proteases in various organs of rats. Participation of proteases in degradation of ornithine aminotransferase in vitro and in vivo.

Homogenates of the muscle layer of rat small intestine irreversibly inactivated endogenous ornithine aminotransferase at 37 degrees C. Addition to the homogenate of coenzymes and the various keto-acids which act as substrate inhibited conversion of the holoenzyme to the apoenzyme and its subsequent degradation. Addition of protease inhibitors including soybean trypsin inhibitor, chymostatin and phenylmethylsulfonyl fluoride almost completely prevented inactivation of he enzyme. Immunological activity decreased during inactivation of the enzyme, but its rate of decrease was much slower than that of loss of enzyme activity. Antigen-antibody precipitates from homogenates containing inactivated enzyme, were separated by electrophoresis on sodium dodecylsulfate-polyacrylamide gels. In this way breakdown products of the enzyme were found, indicating that the enzyme in homogenates was inactivated by limited proteolysis. These results obtained in vitro support our previous suggestion (1975) of a stepwise mechanism for degradation of pyridoxal enzymes.

Animals↗
PubMed1975

Studies on new intracellular proteases in various organs of rat. 1. Purification and comparison of their properties.

1. Specific proteases which inactivate the apo-proteins of many pyridoxal enzymes were found in skeletal muscle, liver and small intestine of rats. The protease from these three organs were purified and their properties were compared. 2. The purified proteases from liver and skeletal muscle appeared homogeneous on acrylamide gel electrophoresis. Two different proteases were separated from small intestine. A homogeneous, crystalline enzyme was obtained from the muscle layer while enzyme from the mucosa was partially purified. 3. They showed substrate specificity for pyridoxal enzymes. Their pH optima were in an alkaline region. They showed activity with the substrate of chymotrypsin, N-acetyl-L-tyrosine ethyl ester, but not with that of trypsin, p-toluenesulfonyl-L-arginine ethyl ester. They were inhibited by pyridoxal phosphate or pyridoxamine phosphate and seryl residues were involved in their active center. 4. The four enzymes differed in the following characters: (a) molecular weights; (b) patterns of elution from a CM-Sephadex column; (c) rates of inactivation of substrate enzymes; (d) rates of cleavage of N-acetyl-L-tyrosine ethyl ester; (e) reactivities with antiserum against the enzyme from the muscle layer of small intestine; (f) specific activities. 5. The amino acid composition and effect of chemical modifications of the crystalline enzyme from the muscle layer of small intestine were examined to elucidate its active sites and mode of action. Serine and histidine residues were found to be essential for protease activity. A tyrosine residue was also necessary for activity. Modifications of its sulfhydryl group, amino residues and carboxyl group had no effect on its activity.

Amino Acids↗
PubMed1975

Studies on new intracellular proteases in various organs of rat. 2. Mode of limited proteolysis.

1. The mechanism of proteolysis of ornithine transaminase apoenzyme II by group-specific protease and the relation between the confirmations of ornithine transaminase and its susceptibility to group-specific protease were studied to elucidate the mode of action of the protease. 2. Differences in the conformations of ornithine transaminase apoenzyme II, molecular weight 67000, and ornithine transaminase holoenzyme, molecular weight 140000, were shown by studies on difference spectra produced by various concentrations of ethylene glycol. Increase of the titratable sulfhydryl groups on resolution of the coenzyme from ornithine transaminase also supports this finding. These results are consistent with the facts that the apoenzyme was sensitive to group-specific protease, while the holoenzyme was not. 3. Kinetics studies showed that ornithine transaminase apoenzyme II was degraded by limited proteolysis. Reaction of the native enzyme with group-specific protease resulted in a nick in the enzyme molecule with formation of one homogeneous large product and small peptides. The large product was not degraded further. The large product was indistinguishable from native ornithine transaminase apoenzyme II in various properties including its elution volume on gel filtration, its mobility on disc electrophoresis, its antigenicity, its estimated number of exposed tryptophan residues, and its titratable number of sulfhydryl groups. But unlike the apoenzyme the product did not show tetramerization with coenzyme or catalytic activity, although it retained the ability to bind with coenzyme and had the same number of bound pyridoxal phosphate as the native ornithine transaminase molecule. Thus, native ornithine transaminase apoenzyme II was degraded by limited proteolysis. Unfolded enzyme, denatured by 8 M urea, was degraded extensively. 4. The initial step of intracellular proteins degradation is discussed on the basis of these results.

Amino Acids↗