Search PubMed⌕ Search

Biomedical subjects

E Kominami

Publications and source records attributed to E Kominami.

At least 181 records · Page 10Linked to original sources
PubMed1987

Molecular cloning and sequencing of cDNA for rat cathepsin L.

A near full-length cDNA for rat cathepsin L was isolated. The deduced protein comprises 334 amino acid residues (Mr 37,685) containing a typical signal sequence (N-terminal 17 residues), pro-peptide (96 residues), and the sequence for mature cathepsin L (221 residues). Rat cathepsin L shows 94% amino acid identity with mouse cysteine proteinase. Amino acid sequence homologies of rat cathepsin L with rat cathepsins H and B are 45 and 25%, respectively. These facts indicate that mouse cysteine proteinase is probably mouse cathepsin L and that cathepsin L is more closely related to cathepsin H than cathepsin B.

Amino Acid Sequence↗
PubMed1987

Autodegradation of lysosomal cysteine proteinases.

Repeated injections of Ep-475, a potent cysteine proteinase inhibitor, into rats caused several-fold increase in the hepatic contents of the lysosomal cysteine proteinases cathepsin B, H and L and in the activities of other lysosomal hydrolases. The rates of degradation of these lysosomal enzymes, estimated by repeated injections of cycloheximide, were found to be retarded in Ep475-treated rats, indicating that lysosomal cysteine proteinases are involved in degradation of lysosomal enzymes including proteinases.

Animals↗
PubMed1987

Formation of mixed disulfide of cystatin-beta in cultured macrophages treated with various oxidants.

Macrophage cell cultures were treated with menadione, zymosan, or phorbol myristate acetate (PMA), and changes in productions of superoxide anion and hydroperoxide, and in glutathione oxidation and S-thiolation of cystatin-beta (formation of a mixed disulfide of cystatin-beta and glutathione) were examined. All three compounds promoted production of superoxide anion and hydroperoxide, but only menadione caused extensive oxidation of glutathione. Menadione caused S-thiolation of cystatin-beta in a dose-dependent fashion, but the other two compounds did not. Removal of menadione promptly reduced the oxidation of glutathione and S-thiolation of cystatin-beta induced by menadione. Inhibition of catalase by aminotriazol caused slight increase in the GSSG content in both menadione- and zymosan-treated cells, but not in S-thiolation of cystatin-beta in zymosan-treated cells. None of the three compounds influenced appreciably the activity of glutathione peroxidase, glutathione reductase, or superoxide dismutase in cultured cells. These results indicate that S-thiolation of cystatin-beta occurs in cells in response to oxidative challenge by menadione but not by zymosan or by the tumor promoter PMA. Dethiolation of cystatin-beta by purified thiol transferase and protein disulfide isomerase in the presence of different concentrations of GSH was examined in vitro. Both enzymes catalyzed dethiolation of cystatin-beta at a much lower level of GSH than that required for the non-enzymatic reaction, suggesting the importance of enzymatic catalysis of S-thiolation and dethiolation of cystatin-beta in cells.

Animals↗
PubMed1987

Activation of the intramyofibral autophagic-lysosomal system in muscular dystrophy.

Skeletal muscles obtained from myopathies with myofiber necrosis, including mdx dystrophic mice, plasmocid-induced myopathy in rats, and patients with Duchenne muscular dystrophy, were examined immunohistochemically with anticathepsin-peroxidase conjugates. Strong reactions for lysosomal cysteine proteinases, which can degrade myofibrillar proteins, were demonstrated in macrophages invading and surrounding the necrotic areas and some degenerative myofibers and also in intramyofibral portions of atrophic fibers of dystrophic mice and humans. Apparently normal and regenerating myofibers did not stain for lysosomal cathepsins. Abnormal increases of cathepsins L and B were seen even in the early stage of plasmocid myopathy and in a 20-day-old young mdx mouse before infiltration of macrophages, suggesting that autodigestion by intramyofibral lysosomal proteinases is an important event before digestion of the necrotic fibers by macrophage proteinases. Activation of the intramyofibral lysosomal system, as in muscular dystrophy, was also observed in distal myopathy with rimmed vacuoles without macrophageal infiltration (Am J Pathol 1986, 122:193-198). Thus, this activation seems to be an important, early response to myocellular damage.

Animals↗
PubMed1986

Involvement of thiol proteases in galactosialidosis.

The activities of Z-Phe-Arg-NMec(ZPA) hydrolase, cathepsin B and cathepsin H and the concentration of endogenous thiol protease inhibitor in fibroblasts from patients with galactosialidosis were found not to be significantly different from those in control fibroblasts. Culture for 5 days with thiol protease inhibitors such as leupeptin, E-64 or Z-Phe-Phe-CHN2 partially restored the beta-galactosidase activity of fibroblasts from patients, but did not affect the beta-galactosidase activity of fibroblasts from control subjects. However, culture with leupeptin, but not other protease inhibitors, increased the ZPA hydrolase and cathepsin B activities of fibroblasts from both patients and controls 2- to 4-fold. Sephadex G-75 chromatography showed that the activity of high molecular weight ZPA hydrolase, which was initially predominant in fibroblasts, decreased markedly during their culture with leupeptin, while the activities of lower molecular weight ZPA hydrolase and cathepsin B increased about 5-fold. These results suggest that high molecular weight ZPA hydrolase, which is presumably cathepsin J, degrades beta-galactosidase, and that the defect in galactosialidosis is impaired protection of beta-galactosidase from degradation.

Cathepsin B↗
PubMed1986

Molecular cloning of human ornithine aminotransferase mRNA.

The isolation and characterization of a cDNA clone for the mRNA of human ornithine aminotransferase (OATase; ornithine-oxo-acid aminotransferase; L-ornithine:2-oxo-acid aminotransferase, EC 2.6.1.13), a nonabundant mitochondrial matrix enzyme that is severely deficient in a hereditary chorioretinal degenerative disease (gyrate atrophy), is described. Human liver, retina, and retinoblastoma (Y79) mRNAs were prepared and tested for the OATase mRNA content by in vitro translation, immunoprecipitation, and NaDodSO4/PAGE. The retinoblastoma cells were found to be expressing this enzyme at a relatively high level. The primary translation product of the OATase mRNA is larger than the pure OATase protein on NaDodSO4/PAGE by approximately equal to 4 kDa, suggesting a precursor protein. lambda gt11 cDNA libraries were prepared from the human mRNAs, and the recombinant clones were immunoscreened as plaques with two different preparations of rabbit anti-human OATase antibodies. A clone (lambda gtRB315) was isolated from the retinoblastoma library that reacts with both of the antibody preparations, and the DNA sequence of its 2.1-kilobase-pair cDNA insert was obtained. An open reading frame consisting of 1371 nucleotides is present in the sequence, and a putative translational initiation methionine codon is identified at position 55. A putative leader sequence consisting of 32 amino acid residues is identified, resulting in a precursor protein of 439 amino acid residues and a molecular mass of 48,534 Da and a mature protein of 407 residues and 45,136 Da. The amino acid sequences of seven tryptic peptides (115 amino acid residues) of the pure human OATase were obtained by microsequencing. When the tryptic peptide and cDNA-derived amino acid sequences were compared, homologies in 111 of 115 residues, including a match of 20 consecutive residues, were observed. An RNA blot hybridization of 32P-labeled OATase cDNA to normal human retina and retinoblastoma mRNAs demonstrated an OATase mRNA species of approximately equal to 2.2 kilobases. The level of OATase mRNA in the normal human retina is approximately equal to 1/100th the level of rhodopsin mRNA and 1/5th to 1/10th the level present in the retinoblastoma cells.

Amino Acid Sequence↗
PubMed1986

Purification and tissue distribution of rat cathepsin L.

Cathepsin L was purified to apparent homogeneity from rat kidney. The molecular weight of the enzyme was estimated to be 30,000, but part of the enzyme was found to consist of two polypeptide chains of Mr 25,000 and 5,000. Antibody against rat kidney cathepsin L did not cross-react with rat cathepsin B or H and detected only cathepsin L in crude rat tissue preparations on immunoblotted sheets. The concentrations of cathepsin L in various rat tissues and peripheral blood cells of rats were determined by a sensitive immunoassay, in which the minimum detectable amount of cathepsin L was 20 pg/assay. The concentration of cathepsin L was found to be highest in the kidneys, where it was more than 3 times higher than in the liver, spleen, lungs, and brain. Nervous tissues, especially the cerebellar cortex, also contained fairly high concentrations of cathepsin L, but the heart, skeletal muscle, and gastrointestinal tract contained low concentrations, as did peripheral blood cells. The cathepsin L content of macrophages was 20% of that of cathepsin B. The concentrations of cathepsin L in lymphocytes, neutrophils, and erythrocytes were 10%, 20%, and less than 0.2%, respectively, of those in resident macrophages.

Animals↗
PubMed1986

Effect of starvation and refeeding on autophagy and heterophagy in rat liver.

Increase in the density of liver lysosomes after leupeptin administration was marked in starved rats but only slight in starved-refed rats. The levels of several intracellular enzymes in the liver lysosome fraction purified from leupeptin-treated rats were about 10 to 30 times more in starved rats than in refed rats. However, there was no difference between the intralysosomal levels of endocytosed FITC-labeled asialofetuin in starved and refed rats, indicating that refeeding after starvation markedly suppressed autophagy but not heterophagy in vivo. Immunohistochemical studies with cathepsin B and asialofetuin Fab'-peroxidase conjugates showed that refeeding after starvation markedly altered the cellular distribution of cathepsin B in the liver, resulting in a linear arrangement of the enzyme only on the periphery of hepatocytes. In contrast, endocytosed asialofetuin was found only in the periphery of hepatocytes of both starved and starved-refed rats. These results indicate that autophagy and heterophagy are regulated by different mechanisms in vivo.

Animals↗
PubMed1986

Abnormal increases of lysosomal cysteinine proteinases in rimmed vacuoles in the skeletal muscle.

Skeletal muscle obtained from a patient with distal myopathy with rimmed vacuole formation (DMRV) was examined by a new direct labeled antibody enzyme method of immunohistochemistry. Abnormal increases of cathepsins B and H, capable of degrading the myofibrillar proteins, were demonstrated to be localized at the site of vacuoles and other intramyofibral portions and not to be associated with concomitant increases of their endogenous inhibitors. Autodigestion by these intramyofibral lysosomal proteinases may be of major importance in focal destruction of myofibers and formation of vacuoles, because no myofiber necrosis with invasion of macrophages, overt myositis, and neuropathy was seen in the muscle. These findings provide the first reasonable explanation of myofibral breakdown and atrophy in DMRV, and should be helpful in further studies on the mechanism of myofibral breakdown in various vacuolar myopathies and other myopathies of so far unknown pathogenesis.

Adult↗
PubMed1985

A new function of kininogens as thiol-proteinase inhibitors: inhibition of papain and cathepsins B, H and L by bovine, rat and human plasma kininogens.

The amidolytic activities of papain and rat liver cathepsins B, H and L were strongly inhibited by high (HMM) and low (LMM) molecular mass kininogens from bovine, human and rat plasmas, and their Ki values were estimated to be in the order of 10(-10) - 10(-11)M for papain and 10(-8) - 10(-9)M for cathepsins. The derivatives of bovine kininogens, HMM kinin-free protein, HMM kinin- and fragment 1 X 2-free protein, and LMM kinin-free protein also showed strong inhibitory activity toward these thiol-proteinases. These results suggest that a reactive site which interacts with thiol-proteinases is contained in the heavy chain portion in kininogens.

Animals↗
PubMed1985

Lysosomal sequestration of cytosolic enzymes and lysosomal thiol cathepsins.

Cytoplasmic proteins are degraded with different half-lives in vivo. Large parts of proteins are believed to be degraded primarily in autophagic vacuoles-lysosomal system. However, the mechanism by which cell proteins are delivered to lysosomes and whether such a process might be selective for certain cell proteins are still unresolved. We examined the mechanism of autophagy with isolated autophagic vacuoles. Administration of leupeptin, a inhibitor of lysosomal thiol proteinases, induced the accumulation of numerous autophagic vacuoles in the liver. Highly purified preparation of autophagic vacuoles was isolated by Percoll density gradient equilibrium fractionation of crude lysosomal fractions. When cytosolic enzyme activities in autophagic vacuoles were measured, tyrosine aminotransferase and tryptophan oxygenase with short half-lives, and lactic dehydrogenase and aspartate aminotransferase with long half-lives were detected at similar ratios of enzymes in autophagic vacuoles/cytosol. During the time that cathepsin B plus L activities in autophagic vacuoles are inhibited by the injection of leupeptin, cytosolic enzymes are being accumulated in autophagic vacuoles suggesting that leupeptin blocks intralysosomal proteolysis, and that cytosolic enzymes are sequestered continuously into autophagosomes. Administration of glucocorticoid, which induces the synthesis of tyrosine aminotransferase, tryptophan oxygenase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Dietary manipulation and administration of insulin, which inhibit the formation of autophagic vacuoles, suppressed completely the accumulation of autophagic vacuoles in liver by administration of leupeptin. Results indicate that there is no selective uptake of cytosolic enzymes into autophagosome. When distribution of lysosomal cathepsin B and L in liver, which are inhibited strongly by leupeptin, was examined immunohistochemically, cathepsin L is found only in hepatocytes, but cathepsin B is localized in sinusoidal cells rather than in hepatocytes, suggesting that cathepsin L plays a most important role in intralysosomal proteolysis in hepatocytes.

Animals↗
PubMed1985

Distribution of cathepsins B and H in rat tissues and peripheral blood cells.

The concentrations of cathepsins B and H in various tissues and peripheral blood cells of rats were determined by means of sensitive immunoassays. The minimum detectable amounts of cathepsins B and H were 30 pg and 20 pg/assay, respectively, and the presence of endogenous thiol proteinase inhibitors did not interfere with the immunoassays. Cathepsin B was found at high levels in the kidney, vagina, spleen, and adrenal gland, and cathepsin H at high levels in the kidney, vagina, liver, lung, and spleen. Low levels of cathepsins B and H were present in the heart, skeletal muscle, and testis. The ratios of cathepsins B and H in various organs were different: the brain and adrenal gland contained much higher levels of cathepsin B than of cathepsin H, whereas the lung and liver contained higher levels of cathepsin H than of cathepsin B. In several organs such as the kidney, spleen, liver, and lungs, the level of cathepsins B plus H was much higher than that of thiol proteinase inhibitors (TPI-alpha + TPI-beta), whereas in tissues containing large amounts of TPI-alpha, such as the skin, esophagus and stomach, the level of inhibitors was higher than that of cathepsins B plus H. Of the peripheral blood cells tested, macrophages had the highest contents of cathepsins B and H, and so their level of cathepsins B plus H was much higher than that of TPI-alpha plus TPI-beta, whereas lymphocytes and neutrophils contained comparable amounts of proteinases and inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Different immunolocalizations of cathepsins B, H, and L in the liver.

Different localizations of cathepsin B, H, and L in normal rat liver were revealed immunohistochemically with anticathepsin Fab'-horseradish peroxidase conjugates. Staining of cathepsin B was strong in the periportal sinusoids, possibly in Kupffer cells; and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of the cytoplasm, possibly representing the peribiliary dense bodies; and weaker in periportal sinusoidal cells, possibly Kupffer cells. Staining of cathepsin L was strongest in centrilobular hepatocytes and weaker in periportal sinusoidal cells, possibly Kupffer cells. These findings, revealed for the first time in the present study, show that the histologic and intracellular localizations of the three cathepsins are different, suggesting that they have different roles in degradation of exogenous and endogenous proteins.

Animals↗