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Biomedical subjects

A Kitahara

Publications and source records attributed to A Kitahara.

At least 37 records · Page 2Linked to original sources
PubMed1986

Two different molecular species of pig calpastatin. Structural and functional relationship between 107 kDa and 68 kDa molecules.

Calpastatin, the inhibitor protein acting specifically on calpain (EC 3.4.22.17; Ca2+-dependent cysteine proteinase), is known to be widely distributed in mammalian and avian cells. Two different molecular species of calpastatin were isolated and purified to homogeneity from pig heart muscle and from pig erythrocytes, and shown to be of 107 kDa and 68 kDa respectively on SDS/polyacrylamide-gel electrophoresis. Both calpastatins had very similar amino acid compositions when expressed as mol per cent of the residues, differed by only 0.1 pH unit in their isoelectric points, and showed immunological cross-reactivity. One molecule of the 107 kDa species could bind approx. 8 calpain molecules, whereas the 68 kDa inhibitor could bind approx. 5 calpain molecules. These findings suggest similar protein structures of the 107 kDa and 68 kDa calpastatins, each being composed of extended multidomains, with unit inhibitor domains aligned along the polypeptide chain of the molecule. The present study does not conclude, however, whether or not the 68 kDa calpastatin found in erythrocytes is a derived product from the 107 kDa species, which is present as such in heart muscle.

Amino Acids↗
PubMed1986

Reversed distribution of calpains and calpastatin in human pituitary gland and selective localization of calpastatin in adrenocorticotropin-producing cells as demonstrated by immunohistochemistry.

The immunohistochemical distribution of Ca2+-dependent cysteine proteinases (calpains I and II) and their endogenous inhibitor calpastatin in normal and adenomatous human pituitary tissue was studied using specific antibodies. The distributions of calpain and calpastatin were dissimilar in human pituitary gland, i.e. ACTH-immunoreactive cells were strongly positive for calpastatin and negative for calpains. PRL-, GH-, FSH-, and TSH-producing cells were negative for calpastatin, but moderately positive for calpains, especially for calpain II, the high Ca2+-requiring form of the enzyme. Similar results were found in pituitary adenoma tissue. These findings indicate that each type of cells producing a specific hormone is equipped with a different balance of the enzyme-inhibitor system involved in the Ca2+-dependent degradation of intracellular proteins.

Adenoma↗
PubMed1985

Selective localization of calpain I (the low-Ca2+-requiring form of Ca2+-dependent cysteine proteinase) in B-cells of human pancreatic islets.

An immunohistochemical study was performed to localize two distinct Ca2+-proteases (low-Ca2+-requiring calpain I and high-Ca2+-requiring calpain II) and their specific inhibitor (calpastatin) in human pancreas using the respective monospecific antibodies. Strongly positive staining by anti-calpain I antibody was found in pancreatic islets, specifically in B-cells, whereas the exocrine pancreatic tissue showed essentially no positive immunostaining. No such specific staining was found with anti-calpain II antibodies or anti-calpastatin antibodies. The results suggest that the Ca2+-dependent proteolysis in B-cells can be triggered by a small rise of the intracellular Ca2+ concentration without serious interference by the endogenous inhibitor.

Calcium↗
PubMed1985

Identification of heterogeneous and microheterogeneous subunits of glutathione S-transferase in rat liver cytosol.

Subunits of multiple molecular forms of dimeric glutathione S-transferase in rat liver cytosol were analyzed by two-dimensional gel electrophoresis (isoelectric focusing/sodium dodecyl sulfate-electrophoresis) followed by staining with Coomassie blue dye. The five subunits, Ya, Yb, Yb', Yc, and Yp (Mr's 26,500, 27,500, 27,500, 28,500, and 26,000, respectively) of seven molecular forms, A2, AC, C2, B2, BL, L2, and GST-P, were identified by comparison of molecular weights and pI values with those of purified molecular forms and by immunoadsorption of the molecular forms in the cytosol as well as those synthesized in vitro using antibodies against the seven forms. Yp is the subunit of placental glutathione S-transferase, GST-P (YpYp), which is markedly increased in carcinogen-treated rat livers [A. Kitahara et al. (1984) Cancer Res. 44, 2698-2703; K. Satoh et al. (1985) Proc. Natl. Acad. Sci. USA 82, 3964-3968]. Microheterogeneity was detectable within Yb, Yb', and Yp subunits, the different forms, termed Yb1, Yb2, Yb'1, Yb'2, and Yp1, Yp2, being similar in size but differing by approx. 0.3 pI unit within each subunit. These microheterogeneous forms were also detectable in the polypeptides translated in vitro in a rabbit reticulocyte lysate translation system from liver poly(A)-containing RNAs, suggesting that they are translatable from distinct mRNAs.

Animals↗
PubMed1985

Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis.

A polypeptide of Mr 26,000 and pI 6.7 that was markedly increased in rat livers bearing hyperplastic nodules (HNs) induced by chemical carcinogens was identified immunochemically as the subunit of neutral glutathione (GSH) transferase (GSHTase; RX:glutathione R-transferase, EC 2.5.1.18; also called GSH S-transferase) purified from placenta (GSHTase-P) and was demonstrated immunohistochemically to be localized in preneoplastic foci and HNs. In the present study, GSHTase-P has been purified from the HN-bearing liver, and the distribution and inducibility have been examined quantitatively using anti-GSHTase-P antibody. Elevation of GSHTase-P in the HN-bearing livers was also confirmed by in vitro translation of mRNAs isolated from the HN-bearing livers. The purified GSHTase-P was homogeneous in size but had two charge isomers on two-dimensional gel electrophoresis. In normal tissues, including liver, placenta, and fetal liver, the protein content of GSHTase-P was generally low but was significantly high in kidney and pancreas. In contrast, the amount of GSHTase-P in HN-bearing livers (primary hepatomas) and transplantable Morris hepatoma 5123D were several 10-fold higher than that in normal liver but were undetectably low in transplantable Yoshida ascites hepatoma AH 130. Different from ordinary drug-metabolizing enzymes, GSHTase-P was uninducible by administration of drugs and carcinogens prior to appearance of the preneoplastic foci and HNs. In addition, species specificity of GSHTase-P was low as it was crossreactive among rat, hamster, and human.

Amino Acids↗
PubMed1985

A protein cross-reacting immunohistochemically with rat glutathione S-transferase placental form as a marker for preneoplasia in Syrian hamster pancreatic and hepatocarcinogenesis.

Immunohistochemical staining using antirat glutathione S-transferase placental form (GST-P) rabbit antibody demonstrated marked binding to cells of putative preneoplastic lesions induced in both the liver and pancreas of Syrian hamsters by dihydroxy-di-n-propylnitrosamine treatment, whereas background normal-appearing tissue was negative. Thus, a protein showing immunological cross-reaction with rat GST-P is also elevated during pancreatic carcinogenesis and hepatocarcinogenesis in the hamster, suggesting its usefulness as a marker with potential relevance to the mechanisms underlying the neoplastic process.

Animals↗
PubMed1984

Intracellular localization of two distinct Ca2+-proteases (calpain I and calpain II) as demonstrated by using discriminative antibodies.

Intracellular localization of two molecular species of calpain (Ca2+-dependent cysteine proteinase) was studied by immunocyto- and histochemical methods employing antibodies strictly monospecific for the respective antigens. Apparent immunological cross-reactivity between the larger subunits of calpain I (low Ca2+-requiring form) and calpain II (high Ca2+-requiring form) was calculated to be 15-17%, and two steps of affinity chromatography were needed to obtain antibodies which can discriminate between the two proteases. Indirect immunofluorescent staining of cultured PK 15 cells revealed diffuse staining of the cytoplasm with both antibodies against calpain I and calpain II. Preincubation with Ca2+-ionophore had no effect on the staining patterns. Sections of porcine kidney were stained by the avidin-biotinylated peroxidase complex method. The proximal and distal tubules and collecting duct were stained, but the glomerulus, macula densa, and vascular vessels were not stained by either anti-calpain I or anti-calpain II antibodies.

Animals↗
PubMed1984

Induction by butylated hydroxyanisole of specific molecular forms of glutathione S-transferase and UDP-glucuronyltransferase and inhibition of development of gamma-glutamyl transpeptidase-positive foci in rat liver.

Effects of an antioxidant, butylated (3-tert-butyl-4-) hydroxyanisole (BHA) on the induction of specific molecular forms of glutathione S-transferase (GST), UDP-glucuronyltransferase (UDP-GT) and other glutathione-related enzymes in rat liver were investigated. The development of gamma-glutamyl transpeptidase (gamma-GTP)-positive foci and hyperplastic nodules induced by diethylnitrosamine, 200 mg/kg i.p., followed by 0.02% N-2-fluorenylacetamide (FAA) in diet plus partial hepatectomy was inhibited by the administration of 0.75% BHA in the FAA-containing diet. Inhibition was reflected in decreased area of gamma-GTP-positive foci which correlated with a decrease in gamma-GTP activity measured biochemically. Under the present experimental conditions, total activities of GSTs, especially that of GST-A form, and of UDP-GTs, especially that of the late fetal form (o-GT), were markedly increased, together with glutathione levels in the whole liver, within one week after BHA administration. Without BHA administration the activities of GST-A and o-GT, as well as glutathione levels, were also increased by FAA treatment, primarily localized within gamma-GTP-positive foci. These results suggest that the induction of specific molecular forms of detoxicating enzymes either in enzyme-altered foci or in the whole liver may play an important role in determining the extent of development of preneoplastic nodules from initiated foci under the short term induction conditions used.

Animals↗
PubMed1984

Large-scale purification of porcine calpain I and calpain II and comparison of proteolytic fragments of their subunits.

Large-scale purification of calpain [Ca2+-dependent cysteine proteinase; EC 3.4.22.17] from porcine tissues is described. The methods used included chromatographies on DEAE-cellulose, Ultrogel AcA 34, Blue Sepharose CL-6B, and DEAE Bio-Gel A which yielded homogeneous enzyme proteins: 27.0 mg of calpain I (low Ca2+-requiring form) from 5 liters of blood with 17,900-fold purification and 57.6 mg of calpain II (high Ca2+-requiring form) from 1.5 kg of kidneys with 5,800-fold purification. Porcine calpains I and II are half-maximally activated at 2.8 microM and 150 microM Ca2+, respectively. They are composed of large and small subunits: Mr 83,000 and 29,000 for calpain I and Mr 80,000 and 29,000 for calpain II. Gel-electrophoretic analysis of the digest with a-chymotrypsin or Staphylococcus aureus V8 protease revealed that the large subunits of calpains I and II are markedly different in structure whereas the small subunits are most likely identical. Mono-specific antibodies directed toward the respective large and small subunits were used for immunoblotting experiments which established not only the identity among several porcine tissues of calpain I but also that of calpain II. several porcine tissues of calpain I but also that of calpain II.

Animals↗
PubMed1984

Enzyme immunoassay of calpain I and calpastatin and its application to the analysis of human erythrocyte hemolysate.

A highly sensitive sandwich enzyme immunoassay for a Ca2+-dependent cysteine proteinase (calpain I) and its specific endogenous inhibitor protein (calpastatin) was developed. The calpain I and calpastatin used as immunogens were purified from human erythrocytes. Anti-calpastatin antisera having sufficiently high titer were obtained only when the immunogen was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay method was principally based on the report by M. Imagawa et al. (1982, J. Appl. Biochem. 4, 41-57), using a specific antibody-coated polystyrene ball and horseradish peroxidase-conjugated Fab' fragment of the antibody. The sensitivity was 0.1 ng of calpain I or calpastatin per assay tube. Starting with 50 microliter of the hemolysate from human erythrocytes, the method permitted direct and simultaneous determination of calpain I and calpastatin, without prior separation of these two enzymatically counteracting components by chromatography. The present method as applied to the erythrocytes from 14 healthy adults gave 120-170 micrograms for calpain I and 164-211 micrograms for calpastatin per gram of hemoglobin, respectively.

Calcium-Binding Proteins↗
PubMed1984

[Effects of krestin and p-aminobenzoic-N-xyloside sodium salt on activities of drug-metabolizing enzymes and glutathione-related enzymes in rat liver].

Effects of krestin (PSK) and p-aminobenzoic-N-xyloside sodium salt (K-247) (both products of Kreha Chemical Co., Japan) on activities of drug-metabolizing enzymes and glutathione (GSH)-related enzymes were investigated in rat liver. When PSK was administered at a dose of 100 mg/kg body weight, ip every day for 7 or 14 days, the action of UDP-glucuronyltransferase (UDP-GT) on o-aminophenol (o-GT) and those of GSH S-transferase (GST) on both 1, 2-dichloro-4-nitrobenzene ( DCNB ) and 1-chloro-2, 4-dinitrobenzene (CDNB) slightly increased together with increased activities of GSH-peroxidase on both H2O2 and cumene hydroperoxide, were as GSH levels were decreased. When PSK or K-247 was administered at 1% in diet for 4 or 8 weeks, o-GT activity and GST activities with both substrates increased on K-247 feeding, while GST activity for CDNB decreased on PSK and K-247 feedings. These changes were statistically significant but very small. The content of P-450 and the activity of gamma-glutamyl transpeptidase changed little in any administration schedules mentioned above.

4-Aminobenzoic Acid↗
PubMed1984

Changes in molecular forms of rat hepatic glutathione S-transferase during chemical hepatocarcinogenesis.

Changes in molecular forms of hepatic cytosolic glutathione S-transferases (GST) during rat chemical hepatocarcinogenesis were investigated. GST activities toward 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene increased with the increased area of gamma-glutamyltranspeptidase-positive foci and hyperplastic nodules induced by diethylnitrosamine followed by 2-acetylaminofluorene plus hepatectomy. Among GSTs with high activities toward 1,2-dichloro-4-nitrobenzene, which were separated by carboxymethyl Sephadex column chromatography, the activity of GST-A ( YbYb ) markedly increased with increased activity towards 1,2-dichloro-4-nitrobenzene in livers bearing foci and nodules and in isolated nodules and hepatomas, while activities of GST-C ( YbYb ') and -D (Yb'Yb') changed little. It was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis that Yb as well as Ya , a subunit of ligandin ( YaYa ) and GST-B ( YaYc ), increased in livers bearing foci and nodules, while Yc as well as Yb' changed little. A new placental GST form (GST-P), which has a subunit molecular weight of 21,500 or 26,000, according to the marker proteins used, and neutral pls of 6.8 and 6.3, is immunologically different from any form of basic GSTs and is very low in normal liver; also, it was markedly induced in livers bearing foci and nodules and in well-differentiated hepatomas but not by short-term administration of drugs such as 2-acetylaminofluorene, in contrast to GST-A and ligandin. These results indicate that GST-A and more especially GST-P could be new preneoplastic marker enzymes for chemical hepatocarcinogenesis.

2-Acetylaminofluorene↗
PubMed1984

The placental form of glutathione S-transferase as a new marker protein for preneoplasia in rat chemical hepatocarcinogenesis.

A neutral form of glutathione S-transferase (GST-P), which has a subunit of 26,000 (or 21,500) daltons and charge isomers with isoelectric points of 6.7 (major), 6.3 and 6.0, was purified from rat placenta cytosol. Immunohistochemical staining using anti-GST-P rabbit antibody demonstrated that GST-P, which is hardly detectable in normal liver, is markedly increased and localized in preneoplastic hepatic lesions such as hyperplastic nodules, indicating that GST-P could be a useful marker proteins for preneoplasia in chemical hepatocarcinogenesis.

Animals↗
PubMed1983

Two distinct Ca2+ proteases (calpain I and calpain II) purified concurrently by the same method from rat kidney.

Two molecular species of calpain (Ca2+-dependent cysteine proteinases) were concurrently purified from rat kidney, both to homogeneity. Calpain I and calpain II having low and high Ca2+ requirements, respectively, were clearly separated on DEAE-cellulose chromatography at pH 7.5, and thereafter they were purified by separate but almost identical procedures which included (NH4)2SO4 fractionation and successive chromatographies on TSK-Gel G 3000 SWG, blue Sepharose CL-6B, and DEAE-Bio-Gel A. The purification folds and activity yields were 6170-fold and 17.8% for calpain I and 4160-fold and 11.9% for calpain II. Ca2+ concentrations for half-maximal activation were 2 microM for calpain I and 200 microM for calpain II. The specific activity of calpain II on casein as the substrate was more than twice higher than that of calpain I. Both enzymes are heterodimers, each composed of 80,000-Da and 25,000-Da subunits. The amino acid compositions of calpain I and calpain II are very similar but not identical. Calpain II is more acidic (pI 4.6) than calpain I (pI 5.3). This paper is the first to describe parallel isolation and characterization of low and high Ca2+-requiring proteases from one single nonmuscular tissue.

Amino Acids↗
PubMed1983

Properties of the increased glutathione S-transferase A form in rat preneoplastic hepatic lesions induced by chemical carcinogens.

Glutathione S-transferase A form (GST-A) is increased markedly in rat preneoplastic hepatic lesions such as hyperplastic nodules induced by diethylnitrosamine followed by administration of N-2-fluorenylacetamide. GST-A was also significantly increased in livers of rats after short-term administration of some drugs. The increased activity and protein content of GST-A were demonstrated by CM-Sephadex C-50 column chromatography as well as by two-dimensional polyacrylamide gel electrophoresis following immuno-affinity column chromatography using antibody against GST-A. Immunologically, GST-A crossreacted strongly with GST-C, weakly with GST-C2, but not with ligandin, GST-B, or GST-AA. It was confirmed by subunit recombination that GST-C is a heterodimer composed of the subunits of homodimers, GST-A and GST-C2.

2-Acetylaminofluorene↗
PubMed1983

Similarity and dissimilarity in subunit structures of calpains I and II from various sources as demonstrated by immunological cross-reactivity.

The structural relationship between calpain I (low Ca2+-requiring) and calpain II (high Ca2+-requiring) molecules and their respective larger (80K) and smaller (30K) subunit proteins of several non-muscular tissues and cells was studied by testing immunological cross-reactivities. In addition to qualitative analyses by a conventional double immunodiffusion method, quantitative data were obtained, for the first time, by enzyme-linked immunosorbent assays using affinity-purified anticalpain I and anti-calpain II immunoglobulins. The enzyme sources included rat kidney, porcine kidney and erythrocytes, and human erythrocytes. It was concluded that the 30K subunits are immunologically almost indistinguishable between calpains, either I or II, not only from the same but also from different sources, while the 80K subunits of different origins are immunologically related to variable extents but always with discrimination between calpain I and calpain II. Similarity of the 30K subunit proteins and dissimilarity of the 80K counterparts were further substantiated by their chromatographic and electrophoretic behaviors.

Animals↗