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Biomedical subjects

Y Kitagawa

Publications and source records attributed to Y Kitagawa.

At least 397 records · Page 22Linked to original sources
PubMed1982

Purification and characterization of two protein kinases from bovine heart mitochondrial membrane.

Two protein kinases (MPK1 and MPK2) were isolated from bovine heart mitochondria. After the solubilization of submitochondrial particles with cholate, these protein kinases were purified by ammonium sulfate precipitation, Sepharose 6B gel filtration, and affinity chromatography on phosvitin-Sepharose. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation of MPK1 contained two major polypeptide bands (40,000 and 36,000 daltons). MPK2 contained one major polypeptide of 34,000 daltons. Under nondenaturing conditions, the molecular weights of MPK1 and MPK2 were estimated to be approximately 250,000 and 70,000-90,000, respectively. MPK1 had a pH optimum at 9.0 and MPK2 at 7.5. Both enzymes required Mg2+ for activity and responded poorly to Mn2+ or Ca2+. Both had similar apparent Km values for ATP and were not affected by either cyclic AMP or Ca2+-calmodulin. MKP1 phosphorylated threonine residues and MPK2 serine residues of casein. With beta casein as substrate, MPK1 was more active than MPK2, whereas with alpha casein, MPK2 was more active than MPK1.

Animals↗
PubMed1982

In situ immunofluorescent visualization of chromosomal transcripts in polytene chromosomes.

The induction and distribution of chromosomal transcripts in the polytene chromosomes of D. melanogaster and D. hydei has been investigated by indirect immunofluorescence using an antiserum directed against DNA/RNA hybrids. The fluorescence was intense and occurred in most of the chromosomal subdivisions when the chromosomes were exposed to denaturing conditions and then allowed to reanneal. The extent of hybrid formation depended both on the extent of DNA denaturation and on the maintenance of RNA integrity. Fluorescence was absent from chromosomes treated with pancreatic RNase before denaturation. The velocity of the chromosomal DNA/RNA hybridization reaction and the effects of the initiation inhibitor of RNA synthesis, DRB, suggest that in order to hybridize the RNA has to be located in its transcriptional compartment. Even though overall patterns of fluorescence seem to be similar during a developmental stage, variations were observed, particularly some correlated with puff induction after ecdysone stimulation.

Animals↗
PubMed1982

Long-term prognosis in ischemic cerebrovascular disease in relation to cerebral blood flow and metabolism.

The correlation between long-term prognosis and cerebral hemodynamics and oxygen consumption was investigated in 46 patients with ischemic supratentorial cerebrovascular disease. Cerebral blood flow (CBF) and cerebral oxygen consumption (CMRO2) were measured by the N2O method 1-6 months (mean 68 days) after disease onset. No significant correlation was observed among CBF, CMRO2 and activities of daily living at the time of measurement. The patients' physical condition was reevaluated by the questionnaire method 2 years or more (mean 53 months) later. The CBF values in patients who were independent after completion of the follow-up period significantly exceeded those of patients dying during the period (P less than 0.05). No correlation was observed in CMRO2. The relationship among CBF, CMRO2 and changes in physical condition during the period was evaluated. Mean CBF values in patients with better prognosis exceeded those of patients with poor prognosis. Notably, CBF values in the "improved" group significantly exceeded those in the "unchanged" or "died" group (P less than 0.05, P less than 0.01). CMRO2 was not significantly correlated with changes of physical condition. These results emphasize the importance of total CBF for long-term prognosis and indicate that flow values, rather than metabolic indices, obtained in the chronic stage can usefully predict the long-term prognosis.

Aged↗
PubMed1982

Comparison of poly(A).poly(dT) and poly(I).poly(dC) as immunogens for the induction of antibodies to RNA-DNA hybrids.

A goat immunized with poly(A).poly(dT) produced three distinct antibody populations. The major one was specific for RNA-DNA hybrids and was purified from precipitates made with poly(I).poly(dC). It also reacted with hybrids of mixed base composition made with E. coli RNA polymerase. The other populations were purified with poly(A).poly(U) or poly(dT). Three rabbits also produced mainly hybrid-specific antibody in response to poly(A) . poly(dT). A goat immunized with poly(I).poly(dC) formed antibodies reactive with poly(I) and others reactive with poly(dC) but none specific for hybrid structure. Three rabbits did not respond to poly(I).poly(dC). Measurements of reactions with anti-inosine sera, thermal denaturation and sensitivity to S1 nuclease indicated that poly(I).poly(dC) is a less stable helix than poly(A).poly(dT) or poly(I).poly(C). Poly(A).poly(dT) is the more suitable synthetic immunogen for the production of hybrid-specific antibodies.

Animals↗
PubMed1982

The separation of three antibody populations from anti-poly(A).poly(U) antibodies elicited in mice or rabbits and antigenic features of poly(A).poly(U)).

Anti-poly(A).poly(U) antibodies in ascitic fluid of DDY mice immunized with poly(A).poly(U)-methylated bovine serum albumin complexes were fractionated into three major antibody populations, Ab-1, Ab-2 and Ab-3, by precipitating with poly(I).poly(C), poly(A).poly(U), and poly(A).2 poly(U), respectively. Antibody population one, Ab-2, reacted with various double-stranded RNAs [poly(I).poly(C), poly(A).poly(U), and rice dwarf virus ribonucleic acid (RDV-RNA)] and poly(A).2 poly(U). Ab-2 reacted with poly(A).poly(U) and poly(A).2 poly(U). Although both Ab-1 and Ab-2 reacted with poly(A).poly(U), the two populations were distinguishable by their different reactivities against chemically modified antigens and oligonucleotides. In contrast to Ab-2, acetylation at the furanose 2'-position of poly(U) resulted in a dramatic decrease in the complement fixation reactivity of Ab-2. Also, Ab-2 was capable of binding with complexes of hexa- to heptaadenylates and poly(U), whereas Ab-1 required oligoadenylates of longer chain lengths (9-10 chain length) for binding. Therefore, it appears that poly(A).poly(U) possesses unique antigenic determinants which are recognizable only by Ab-2, in addition to those determinants which are common to a variety of double-stranded RNAs.

Animals↗
PubMed1982

Perturbation of phospholipid metabolism by erucic acid in male Sprague-Dawley rat heart.

Erucic acid was incorporated into cardiac phosphatidylserine and hepatic and cardiac sphingomyelin of male Sprague-Dawley rat. The fatty acid compositions of mitochondrial and microsomal phospholipids were similar in both liver and heart. The effect of a low fat diet and a diet containing erucic acid on the fatty acid composition of mitochondrial and microsomal phospholipids was also similar, except for the effect on sphingomyelin. However, the diet containing erucic acid influenced the metabolism of phosphatidylcholine of the heart but not of the liver, indicating that the turnover of 1-stearoyl-2-arachidonoyl phosphatidylcholine in the heart was inhibited by the diet containing erucic acid. On the other hand, the proportion of erucic acid in the free fatty acid was higher in the heart than in the liver.

Animals↗
PubMed1980

The specific binding of parotin on duct cells of human parotid gland.

The relationship between parotin and human parotid gland was investigated using indirect technique of enzyme antibody method. Normal rabbit IgG has non-specific affinity to duct cells of human parotid gland, but it was suppressed by adding bovine serum albumin. Anti-parotin rabbit IgG has no reaction products on the sections of adult human parotid gland. When the sections, initially incubated with parotin solution in PBS, were treated with anti-parotin rabbit IgG containing bovine serum albumin, and then HRPO-labeled anti-rabbit gamma globulin antibodies (swine IgG), intercalated and striated ducts excepting excretory ducts were stained with diaminobenzidine reaction, but also acinar cells did not. These results indicate that parotin is bound specifically on duct cells of human parotid gland.

3,3'-Diaminobenzidine↗
PubMed1980

Effect of erucic acid on the phospholipid molecular species compositions of the rat heart and liver.

Erucic acid was not incorporated into phosphatidylcholine or phosphatidylethanolamine, but was into diphosphatidylglycerol and sphingomyelin in both the heart and liver of male Wistar rats fed erucic acid for 20 days. A remarkable difference between the heart and liver was found in the molecular species composition of phosphatidylcholine. The 1-stearoyl//2-arachidonoyl species of phosphatidylcholine in the heart was maintained at a higher level in rats fed the erucic acid diet than in rats fed a low fat diet. There was no difference in this molecular species content of cardiac and hepatic phosphatidylethanolamine between rats fed erucic acid and low fat diets. The level of erucic acid incorporated into triacylglycerols and free fatty acids in heart was higher than in the liver.

Animals↗
PubMed1980

Estimation of the in vivo translational activity of rat liver mitochondria without use of an antibiotic.

In order to estimate the in vivo translational activity of mitochondria without using cycloheximide, we developed a selective separation procedure for the mitochondrial translation products. The effects of cycloheximide and isotopic dilution kinetics on the precursor leucine pool indicate that the pH 11.5 insoluble mitochondrial fraction is greatly enriched in mitochondrial translation products.

Animals↗
PubMed1980

Purification of rat liver glycerate kinase and studies of its enzymatic and immunological properties.

Glycerate kinase was purified to near homogeneity from rat liver mitochondria. Sephadex G-100 chromatography and sodium dodecyl sulfate-gel electrophoresis showed that the enzyme is a monomer with a molecular weight of 51,000-56,000. Kinetic studies indicated that the reaction proceeds by the "rapid equilibrium random sequential" mechanism, and the Michaelis constants were found to be 0.032 mM and 0.091 mM for D-glycerate and ATP, respectively. The binding of D-glycerate specifically protected the enzyme from thermal inactivation. The dissociation constant of the enzyme.D-glycerate complex was similar to the Michaelis constant, suggesting that the protective effect of D-glycerate may be caused by the specific binding of the substrate to the active site of the enzyme. The antibody to the enzyme was prepared by immunizing a rabbit with the purified rat liver glycerate kinase. Immunological experiments indicated that the cytosol and mitochondrial enzymes are indistinguishable. It was also found by immunological studies that the increase in the cytosol and mitochondrial enzyme activity depending on dietary protein intake was proportional to the increase in enzyme protein. These results support our proposal (Kitagawa, Y., Katayama, H., & Sugimoto, E. (1979) Biochim. Biophys. Acta 582, 260-275) that cytosol and mitochondrial glycerate kinase arise from a common translation product and that dietary protein regulates the distribution of glycerate kinase to the cytosol and mitochondria.

Animals↗
PubMed1980

Demonstrations of the production of specific antibodies to poly(I).poly(C) in rabbits.

The rabbit antiserum against poly(I).poly(C) purified by hydroxyapatite column chromatography contained three distinct antibodies. They were fractionated into three antibody populations by a series of precipitations (with poly(A).poly(U), poly(I), and poly(I).poly(C)) and their specificities were examined by quantitative complement fixation, double diffusion tests and radioimmunoassay. The first population was common to the double helical structure of double-stranded RNAs. The second was specific for poly(I) and the third was specific for poly(I).poly(C). These studies demonstrated that specific antibodies exclusively reactive with poly(I).poly(C) existed in the rabbit antiserum against poly(I).poly(C).

Animals↗
PubMed1979

Identity of mitochondrial and cytosolic glycerate kinases in rat liver and regulation of their intracellular localization by dietary protein.

Glycerate kinase (ATP: D-glycerate 2-phosphotransferase EC 2.7.1.31) is a key enzyme of glyconeogenesis from serine via hydroxypyruvate. A differential centrifugation of rat liver homogenate and an analysis of the particle fraction by sucrose density gradient centrifugation indicated that 72% and 26% of glycerate kinase are present in mitochondria and cytosol, respectively. A study on the intramitochondrial localization of the enzyme suggested that the mitochondrial glycerate kinase was present in inner membrane and/or matrix. It was found that dietary protein selectively induced mitochondrial glycerate kinase. This result suggested that mitochondrial glycerate kinase had a physiological function for gluconeogenesis from serin. However, the metabolic significance of the cytoplasmic enzyme was still unclear. The properties of solubilized-mitochondrial and cytosolic glycerate kinases were compared. However, no difference between the two enzymes could be found in the kinetic properties, thermal stability, molecular size or electrochemical properties. These results suggested that both enzymes originate from common genetic information. In order to elucidate the regulatory mechanism of the intracellular distribution of glycerate kinase in rat liver, the responses of mitochondrial and cytosolic glycerate kinases to an alteration of dietary protein were studied. The result suggested that an alteration of dietary protein content may regulate the distribution and the translocation of glycerate kinase to mitochondria and cytosol as well as the total amount of glycerate kinase.

Animals↗
PubMed1979

Possibility of mitochondrial-cytosolic cooperation in gluconeogenesis from serine via hydroxypyruvate.

Intracellular localization of D-glycerate dehydrogenase (D-glycerate: NAD+ oxidoreductase, EC 1.1.1.29), one of the enzymes of the pathway for gluconeogenesis from serine via hydroxypyruvate, was studied by differential centrifugation. Almost all enzyme activity was found in cytosol. Since the major activities of two other enzymes, serine: pyruvate aminotransferase (EC 2.6.1.51) and glycerate kinase (ATP: D-glycerate 2-phosphotransferase, EC 2.7.1.31), of the pathway via hydroxypyruvate are localized in mitochondrial inner membrane and/or matrix, the possible localization of D-glycerate dehydrogenase in mitochondria was examined. Detailed analysis of mitochondrial fraction prepared by differential centrifugation indicated that rat liver mitochondria do not contain any D-glycerate dehydrogenase activity. Based on these results, a cooperative connection between mitochondria and cytosol in gluconeogenesis from serine via hydroxypyruvate is proposed. Possible mechanisms for transport of intermediates of the pathway via hydroxypyruvate across the mitochondrial membranes are also discussed.

Animals↗
PubMed1979

Mitochondrial and cytosolic localization of a single glycerate kinase in rat kidney cortex.

The distribution of glycerate kinase [ATP:D-glycerate 2-phosphotransferase, EC 2.7.1.31] in kidney was studied. This enzyme was found to be present in the renal cortex. By differential centrifugation of the homogenate and sucrose density gradient analysis, it was found that 42% and 60% of the renal glycerate kinase were localized in the cytosol and mitochondria, respectively. The mitochondrial enzyme appeared to be present in the inner membrane and/or matrix. No difference was found between the solubilized-mitochondrial and cytosolic glycerate kinase as regards kinetic properties, thermal stability, electrochemical properties, and molecular size. Immunochemical identity of these enzymes was demonstrated using a rabbit antibody against mitochondrial glycerate kinase purified from rat liver. Although the hepatic enzyme was induced by dietary protein (Kitagawa, Y., Katayama, H., & Sugimoto, E. [1979] Biochim. Biophys. Acta 582, 260--275), the renal enzyme in mitochondria and cytosol was not affected by dietary protein. These results on renal glycerate kinase are compared with those for the hepatic enzyme, and the regulatory mechanism for intracellular distribution of the enzymes is discussed.

Animals↗