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H Kanoh

Publications and source records attributed to H Kanoh.

At least 109 records · Page 6Linked to original sources
PubMed1988

Occurrence of immunoreactive 80 kDa and non-immunoreactive diacylglycerol kinases in different pig tissues.

We surveyed diacylglycerol kinase in different pig tissues by using rabbit antibody immunospecific to the brain 80 kDa enzyme [Kanoh, Iwata, Ono & Suzuki (1986) J. Biol. Chem. 261, 5597-5602]. Among the other tissues examined, the immunoreactive 80 kDa enzyme was found only in the thymus and, to a much lesser extent, in the spleen, although this enzyme species was widely distributed in a variety of brain regions. Other tissues such as platelets, kidney, heart and liver contained little, if any, immunoreactive enzymes. Gel filtration of cytosolic enzymes from several tissues revealed the presence of three major activity peaks, apparently corresponding to 280, 120 and 80 kDa. Thymus and spleen contained the immunoreactive 80 kDa species together with non-immunoreactive 280 kDa enzyme. In the case of platelets, the kinase consisted almost exclusively of non-immunoreactive 120 kDa species with some 280 kDa enzyme. In an attempt to characterize the different kinase forms, the thymus enzyme was chosen for further studies because of its high activity. No immunoreactive proteins were detected in Western-blot analysis when the 280 kDa enzyme was solvent-extracted, proteinase-treated or preincubated in the presence of Ca2+. In comparison with the 80 kDa species, the 280 kDa enzyme was much more heat-stable and less dependent on deoxycholate in the assay mixture. Although the purification of different forms of the kinase is required to confirm the presence of isoenzymes, the results show that there exist several immunologically distinct diacylglycerol kinase species.

Animals↗
PubMed1987

Effects of CuCl2 on the dimer-tetramer equilibrium of concanavalin A.

Tetrameric concanavalin A at neutral pH dissociated into the dimer when CuCl2 was added in a concentration range comparable to protomer concentration. This effect of CuCl2 was largely suppressed when NaCl concentration was increased. Neither the conformation of the protein nor its binding activity to 4-methylumbelliferyl alpha-D-mannopyranoside was affected on addition of CuCl2.

Concanavalin A↗
PubMed1986

Phosphorylation of pig brain diacylglycerol kinase by endogenous protein kinase.

Pig brain diacylglycerol kinase did not catalyze autophosphorylation. However, the kinase was phosphorylated on serine, when immunoprecipitated from the partially purified enzyme preparation preincubated with Mg2+ and [gamma-32P]ATP. The action of the endogenous protein kinase phosphorylating diacylglycerol kinase was independent of cyclic nucleotides and Ca2+, and became maximum at pH 5.5. Although the extent of enzyme phosphorylation was limited (maximally about 0.25 mol Pi incorporated per mol kinase), the results show that diacylglycerol kinase can be a phosphoprotein.

Adenosine Triphosphate↗
PubMed1986

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain.

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.

Animals↗
PubMed1985

Arachidonic acid metabolism in cultured mouse keratinocytes.

We attempted to characterize the general features of arachidonate metabolism in cultured mouse keratinocytes. The cells labeled with [3H]arachidonate were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), ionophore A23187, and fetal bovine serum (FBS). Common to the three substances, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine almost equally served as sources of arachidonate liberated by the action of phospholipase A2. The stimulation of phospholipase A2 action was observed in the order of A23187 greater than FBS greater than TPA. When stimulated by TPA or A23187, the radioactivity released into the extracellular medium was mostly found in prostaglandin (PG) E2. Formation of other PGs and hydroxyeicosatetraenoate (HETE) was extremely limited. In the case of stimulation by FBS, however, the released radioactivity was mainly associated with non-converted arachidonate. FBS also inhibited the TPA- and A23187-induced conversion of arachidonate to PGE2. Phospholipid degradation induced by the three stimulators was similarly dependent on extracellular Ca2+. The stimulation by FBS and A23187 was suppressed by calmodulin antagonists, though the effect of A23187 was much more sensitive to the antagonists when compared to that of FBS. We observed more than additive effects of the three stimulators when tested together.

Animals↗
PubMed1984

Utilization of diacylglycerol in phospholipid bilayers by pig brain diacylglycerol kinase and Rhizopus arrhizus lipase.

Diacylglycerol kinase purified from pig brain cytosol could use sonication-dispersed diacylglycerol in the presence of its activator, phosphatidylcholine vesicles. However, the kinase failed to significantly use diacylglycerol cosonicated with phosphatidylcholine. Similarly, the kinase could not use diacylglycerol generated in microsomes by the back reaction of diacylglycerol choline phosphotransferase, though phospholipase C treatment of microsomes yielded effective substrate for the kinase. In order to elucidate the mechanism of these discrepant findings, we studied the activity of the purified kinase and Rhizopus arrhizus lipase utilizing dioleoylglycerol incorporated into various phospholipid vesicles. The inaccessibility of diacylglycerol contained in phospholipid vesicles was observed similarly for the two different enzymes. We considered that the apparent enzymic latency of diacylglycerol could be best accounted for by an extremely limited solubility of diacylglycerol in the outer leaflet of phospholipid bilayers. The experimental bases for this interpretation are: 1) diacylglycerol cosonicated with dihexanoyl phosphatidylcholine was exceptionally effective as substrate for the kinase; 2) the enzyme activities with cosonicated and separately sonicated lipids became similar when bile salts were present; 3) both enzymes could use diacylglycerol generated on phosphatidylcholine vesicles by a limited phospholipase C hydrolysis; and 4) phosphatidylcholine diacylglycerol vesicles at widely different molar ratios (from 1:0.014 to 1:0.2) were similarly ineffective as substrate for both enzymes.

Animals↗
PubMed1984

[Laboratory and clinical studies of cefotiam in digestive diseases].

Experimental and clinical studies on cefotiam (CTM), a new synthetic cephalosporin, were performed in digestive diseases. Among the 7 cases of 'cholelithiasis and cholangitis', 6 cases (86%) showed better response than "effective". Among the 9 cases of infections except 'cholelithiasis and cholangitis' which had no connection with cancers, 6 cases (67%) showed better response than "effective". Among the 15 cases of 'infections following advanced cancers', 7 cases (47%) showed better response than "effective". This inferior result was due to the relation to factors coming from cancers. CTM was effective in not only infections of the aged but that which resisted antibiotics of penicillins and the other cephems. Sensitivity of E. coli, Klebsiella and S. aureus was made a comparative study of by MIC and disc test. It was found as a result that CTM had the equal or superior antimicrobial activity to other antibiotics of cephems, penicillins and aminoglycosides.

Adult↗
PubMed1983

Deacylation of ceramide, triacylglycerol and phospholipids in guinea pig epidermal cells.

The epidermal cells obtained by mild trypsinization of guinea pig skin were first labeled with [3H]palmitate. Interestingly, free ceramide was one of the major radioactive products. The radioactivity of ceramide was located exclusively in the N-acyl moiety, and palmitate was incorporated as such, without detectable chain modification. Sphingomyelin and glucosylceramide were not significantly labeled in spite of the active synthesis of ceramide. In order to investigate the action of lipolytic enzymes, the labeled cells were chased in a tracer-free medium. Bovine serum albumin (or fetal calf serum) and ionophore A23187 markedly stimulated the deacylation of lipids. The effects of the two stimulators were, however, distinctly different from each other. Bovine serum albumin (or fetal calf serum) caused principally the deacylation of triacylglycerol and ceramide, with little effect on that of phospholipids. In contrast, the effect of the ionophore was confined to phospholipid deacylation. In the presence of the ionophore, phosphatidylcholine and phosphatidylethanolamine were degraded to a similar extent. Palmitate was cleaved from both C-1 and C-2 positions of phosphatidylcholine. The results suggest that the lipolytic systems of the epidermal cells can respond to extracellular stimuli, and that the action of triacylglycerol lipase and ceramidase is controlled differently from that of phospholipases.

Acylation↗
PubMed1983

Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies.

Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.

Animals↗
PubMed1982

Characterization of biochemical properties of melanosomes for structural and functional differentiation: analysis of the compositions of lipids and proteins in melanosomes and their subfractions.

Two types of melanosomes were isolated from B16 and Harding Passey (HP) mouse melanomas to elucidate whether there are any features of melanosomal lipids and proteins related to the differences in morphology of melanosomes and in the biological activities of melanoma cells. Biochemical analyses were made to clarify (a) the lipid components of the 2 melanosomes and (b) the lipid and polypeptide compositions of their subfractions, i.e., the outer surface and inner core, resolved by a detergent, Brij-35, separately. We found (a) that the lipid contents in B16 melanosomes were much higher than those in HP, (b) that the B16 and HP melanosomes could be fractionated into a phospholipid-rich outer surface and phospholipid-poor core, (c) that both outer surface and core subfractions of HP were distinct from the corresponding subfractions of B16 with respect to phospholipid contents, (d) that the outer surface of B16 and HP revealed a polypeptide composition similar to each other, although the protein contents of the outer surface were much lower than those of core and (e) that the total melanosomes showed a marked difference in polypeptides between B16 and HP. In addition, both B16 and HP melanosomes revealed the alteration of lipid compositions, e.g., fatty acid acyl chain, similar to that observed during malignant transformation. There was not, however, any significant difference in acyl compositions between B16 and HP. It is likely that melanosomal lipid and protein not only affect the different morphogenesis of melanosomes but also reflect the different biological activities of whole cells, and that lipids, primarily located in the outer surface, regulate the functional aspects of melanosomes.

Animals↗
PubMed1981

Incorporation of radioactive free fatty acids into glycerolipids of subcellular fractions from human tonsil lymphocytes.

Human tonsil lymphocytes were cultured with phytohemagglutinin (PHA) for 1-72 hr. Throughout the culture periods studied, PHA stimulated similarly the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine from radioactive free fatty acids. There was no significant difference in the stimulation of cellular uptake of different labeled fatty acids tested (palmitic, stearic, oleic and arachidonic acids). Human tonsil lymphocytes were shown to be composed of heterogeneous cell populations with respect to the activities of cellular uptake of free fatty acids. The cells were subfractionated after being labeled with radioactive fatty acids for 6 hr. We found that the light and heavy plasma membranes, which are known to occur in human tonsil lymphocytes, are distinctly different from each other with respect to the labeling patterns of glycerolipids and also to the response to PHA. In the light plasma membranes, the labeling of phospholipids relative to that of triacylglycerol was much less than that of heavy plasma membranes and other organelles, though radioactivity of phosphatidylcholine per mg protein of light plasma membranes was the highest of any other organelles. Furthermore, the stimulation of phosphatidylcholine synthesis by PHA appeared most markedly in the light plasma membranes in comparison to other subcellular fractions.

Cells, Cultured↗
PubMed1980

The role of the de novo synthetic pathway in forming molecular species of phospholipids in resting lymphocytes from human tonsils.

Phosphatidylcholine of resting human tonsil lymphocytes contained dipalmitoyl (18 mol%), 1-oleoyl,2-palmitoyl (11 mol%) and dioleoyl (5 mol%) species. 1-Oleoyl and 1-linoleoyl species were also detected in other more highly unsaturated phosphatidylcholine species. The features of phospholipid synthesis in lymphocytes were investigated by incubating cells with radiolabeled precursors. The de novo synthesis of phospholipids occurring in lymphocytes was estimated to be physiologically important, in particular for supplying dipalmitoylglycerophosphocholine and highly unsaturated phosphatidylethanolamine. It was also suggested that diacylglycerol(s) not originating from glycerophosphate is (are) involved in the synthesis of tetraenoic phospholipids. From radioactive palmitic and oleic acids were actively synthesized dipalmitoyl, dioleoyl and 1-oleoyl,2-palmitoyl species of diacylglycerol. The mode of diacylglycerol synthesis was reflected upon phosphatidylcholine formation. The formed polyunsaturated phosphatidylethanolamine was also found to contain 1-oleoyl or 2-palmitoyl species. Radioactive linoleic and arachidonic acids were incorporated predominantly into the C-1 position of diacylglycerol, whereas the majority of the formed phospholipids was of 2-linoleoyl or 2-arachidonoyl species. Labeled stearic acid was exclusively esterified to the C-1 position of the glycerolipids. However, the labeling pattern of molecular species by stearate was considerably deviated from that observed with labeled palmitate. These results indicate that the de novo synthetic pathway operating lymphocytes is primarily responsible for forming 1-unsaturated type of phospholipids. The synthesis of 1-saturated,2-unsaturated species appeared to be due to remodeling of once-formed phospholipids.

Chemical Phenomena↗
PubMed1979

Effect of even- and odd-numbered saturated fatty acids on glycerolipid synthesis in rat liver slices.

Effect of even- and odd-numbered saturated fatty acids, ranging from lauric to stearic acids, was studied on the de novo synthesis of glycerolipids in rat liver slices. For all fatty acids tested, a marked synthesis of saturated glycerolipids was observed except for phosphatidylethanolamine. When compared at the fixed concentration (2 mM), myristic acid caused a peak synthesis of saturated glycerolipids, and the presence of longer or shorter even- and odd-numbered fatty acids resulted in their lesser formation. The formation of saturated species of triacylglycerol and phosphatidylcholine closely followed the mode of synthesis of saturated diacylglycerols, though dipentadecanoyl-and dipalmitoylglycerols appeared to be less converted to the corresponding saturated triacylglycerols in comparison to the other saturated diacylglycerols. Very little formation of saturated diacylglycerols occurred when lauric, tridecanoic and stearic acids were tested. The majority of lauric and tridecanoic acids incorporated into saturated diacylglycerols was shown to be chain-elongated prior to esterification.

Animals↗