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Biomedical subjects

N Katunuma

Publications and source records attributed to N Katunuma.

At least 253 records · Page 14Linked to original sources
PubMed1978

Selective cleavage of peptide bonds by a serine protease from rat skeletal muscle.

The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-Glu, were not susceptible to another serine protease from small intestine (SI-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the SI-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the SI-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the SI-protease.

Amino Acids↗
PubMed1978

Purification and properties of a new cathepsin from rat liver.

1) A lysosomal protease, a new cathepsin that inactivates glucose-6-phosphate dehydrogenase [EC 1.1.1.49] and some other enzymes and differs from cathepsin B [EC 3.4.22.1] was purified about 2,200-fold from crude extracts of rat liver by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, and DEAE Sephadex and CM-Sephadex column chromatographies. 2) The new cathepsin was markedly activated by the thiol-reagent, 2-mercaptoethanol and inhibited by monoiodoacetate. 3) The molecular weight of the new cathepsin was found by Sephadex G-75 column chromatography to be 22,000, which is smaller than that of cathepsin B. 4) The optimum pH of the enzyme for inactivation of glucose-6-phosphate dehydrogenase was pH 5.0--5.5. The enzyme was unstable in alkali and on heat treatment. 5) The rates of inactivation of glucose-6-phosphate dehydrogenase, apo-ornithine aminotransferase [EC 2.6.1.13], apo-tyrosine aminotransferase [EC 2.6.1.5], apo-cystathionase [EC 4.4.1.1], glucokinase [EC 2.7.1.2], glyceraldehyde-3-phosphate dehydrogenase [EC 1.2.1.12], and malate dehydrogenase [EC 1.1.1.37] by the new cathepsin were higher than those by cathepsin B. However aldolase [EC 4.1.2.13] was inactivated more rapidly by cathepsin B than by the new cathepsin. Lactate dehydrogenase [EC 1.1.1.27], glutamate dehydrogenase [EC 1.4.1.2] and alcohol dehydrogenase [EC 1.1.1.1] were not inactivated by either cathepsin. Unlike cathepsin B, the new cathepsin scarcely hydrolyzes N-substituted derivatives of arginine.

Animals↗
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↗
PubMed1978

Mode of binding of pyridoxal 5'-phosphate in rat liver ornithine aminotransferase.

1. Pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate were effective for the association of apo-form II of ornithine aminotransferase [EC 2.6.1.13]; whereas other B6 derivatives, such as pyridoxal, pyridoxamine, pyridoxine and pyridoxine 5'-phosphate, had no effect on form II of this apoenzyme. 2. The pyridoxal 5'-phosphate contents of the native enzyme, and reconstituted forms I and II were determined by two different methods to be 1.5 moles, 2.5 moles and 3.3 moles of pyridoxal 5'-phosphate/mole of enzyme, respectively.

Animals↗
PubMed1977

Studies on the structure of rat liver ornithine aminotransferase.

Two distinct types of apoenzyme from native ornithine aminotransferase [EC 2.6.1.13], named forms I and II, have been found to be composed of a single type of subunit by means of Sephadex G-100 column chromatography in the presence of sodium dodecylsulfate, DE 52 cellulose column chromatography in the presence of 8 M urea and urea-polyacrylamide gel disc electrophoresis. The amino-terminal residues of both apoenzymes were blocked, but several amino acids in sequence from the carboxylterminals were identical and the following sequence was found in both: Phe-Ser-Leu-IIe-Ala-(Val)-. In the native enzyme, 4 SH groups out of a total 20 SH groups/tetramer could be titrated without loss of enzyme activity, while in the apo-form I, 4 SH groups out of a total 20 SH groups/tetramer could be titrated with 80% loss of activity. In the apo-form II, in contrast, 6 SH groups out of a total 10 SH groups/dimer could be titrated and titration of the first 2 SH groups caused 100% loss of activity.

Amino Acids↗
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↗
PubMed1977

Biochemical and histocytochemical studies on response of ammonia-producing enzymes for nh4cl-induced acidosis.

NH4Cl-induced acidosis in rats resulted in renal enlargement and increase in activities of phosphate-dependent glutaminase and glutamic dehydrogenase. The renal enlargement was associated with protein synthesis but not deoxyribonucleic acid synthesis. In control rats histochemical activity of glutamic dehydrogenase was seen dominantly in the proximal straight tubule. In acidotic rats high activity was noted in the proximal convoluted tubule as well as in the proximal straight tubule. By electron microscopy reaction product was in mitochondria. The results suggest that urine ammonia is produced in mitochondria of epithelial cells in the proximal straight tubule in both normal and acidotic rats. Increased enzyme activity in acidotic rats is largely associated with epithelial cells of the proximal convoluted tubule.

Acidosis↗
PubMed1977

Establishment of a clonal strain of hepatoma cells derived from Morris hepatoma 8999.

A new line of tissue culture cells derived from a slow-growing hepatoma 8999 was established and named 8999C. The isolation method, growth pattern, and morphology of the 8999C cells are described. Several hepatic enzyme activities in 8999C cells were compared to those in the original hepatoma 8999. The ornithine aminotransferase (EC 2.6.1.13), tyrosine aminotransferase (EC 2.6.1.5), and arginase (EC 3.5.3.1) activities in the 8999C cells were one-third, one-tenth, and one-hundredth of those of the respective activities in the original hepatoma 8999, and mitochondrial serine protease, which has much higher activity in hepatoma 8999 than in normal liver cells, was not detected in 8999C cells. Tyrosine aminotransferase in 8999C cells was induced by dexamethasone but not by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate or insulin. Unlike in hepatoma 8999, glucocorticoid did not induce arginase in 8999C cells.

Arginase↗
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↗