Biomedical subjects

H Ebata

Publications and source records attributed to H Ebata.

At least 37 records · Page 2Linked to original sources

PubMed1990

Calcium binding to extracellular sites of skeletal muscle calcium channels regulates dihydropyridine binding.

The binding of dihydropyridine (PN200-110) to skeletal muscle microsomes (which were 84% sealed inside-out vesicles) was not influenced by the addition of calcium or magnesium nor by addition of their chelators (EDTA or EGTA) unless the vesicles were pretreated with the calcium-magnesium ionophore A23187 and EDTA to remove entrapped cations. Separation of inside-out vesicles from right-side-out vesicles by wheat germ agglutinin chromatography revealed that only the right-side-out vesicles exhibited a calcium-, magnesium-, and chelator-dependent binding of PN200-110. Dihydropyridine binding to cardiac sarcolemma membranes (which were 46% inside-out) and to solubilized skeletal muscle membranes was inhibited by EDTA and could be fully restored by 10 microM calcium or 1 mM magnesium. Calcium increased PN200-110 binding to partially purified rabbit skeletal muscle calcium channels from 3.9 pmol/mg protein to 25.5 pmol/mg protein with a pK0.5 = 6.57 +/- 0.059 and a Hill coefficient of 0.56 +/- 0.04. Magnesium increased binding from 0.7 pmol/mg protein to 16.8 pmol/mg protein with a pK0.5 = 3.88 +/- 0.085 and a Hill coefficient of 0.68 +/- 0.074. These studies suggest that calcium binding to high affinity sites or magnesium binding to low affinity sites on the extracellular side of skeletal muscle T-tubule calcium channels regulates dihydropyridine binding. Further, similar calcium and magnesium binding sites exist on the cardiac calcium channel and serve to allosterically regulate dihydropyridine binding.

Animals↗

PubMed1989

Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes.

Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 mumol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1'-enyl-sn-glycero-3-phosphocholine or -ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl-sn-glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent Km values of 5.5 and 42 microM for 1-alk-1'-enyl-sn-glycero-3-phosphocholine and 1-alk-1'-enyl-sn-glycero-3-phosphoethanolamine, respectively. The Vmax values were 11.7 and 13.6 mumol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15,200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca2+, Mg2+, Mn2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p-chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37 degrees C. Activity of the purified enzyme, destroyed by freezing at -20 degrees C, was preserved if stored at this temperature in the presence of 300-600 microM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, facilitated this purification. This is the first reported purification of alkenylhydrolase.

Animals↗

PubMed1988

Solubilization and reconstitution of voltage-dependent calcium channel from bovine cardiac muscle. Ca2+ influx assay using the fluorescent dye Quin2.

Highly purified sarcolemmal membranes, prepared from fresh bovine heart left ventricle, were solubilized by n-octyl beta-D-glucopyranoside and reconstituted into proteoliposomes with soybean phospholipids by the detergent-dialysis method. Ca2+ flux into the proteoliposomes was determined using the fluorescent probe Quin2. A membrane potential (negative in the proteoliposome interior) that was created by K+ diffusion mediated by valinomycin accelerated the Ca2+ influx. The voltage-dependent Ca2+ influx was dependent on pretreatment of the sarcolemmal membranes with Bay K 8644 and was inhibited by various calcium antagonists including nicardipine (K0.5 = 4.5.10(-7) M), verapamil (K0.5 = 9.2.10(-9) M), diltiazem (K0.5 = 26.10(-8) M) and omega-conotoxin (K0.5 = 9.5.10(-9) M).

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1988

A study of liver regeneration using fetal rat liver tissue transplanted into the spleen.

The liver morphology of fetal hepatic tissue transplanted into an ectopic location was investigated over one year period. Fetal liver fragments prepared from a maternal rat on the 18th or 19th day of pregnancy were injected into the splenic parenchyma of syngeneic rats using a 21 gauge needle. Histologically, the fetal liver did not essentially show any apparent lobular architecture or cord structure. The transplanted fetal hepatic tissues survived and formed hepatic cords in the spleen instead of undergoing degeneration and necrosis. Three characteristic features became complete during the 4 weeks following transplantation, namely; clumps of hepatocytes with obvious hepatic cords and sinusoids, markedly proliferating bile ducts and proliferating individual hepatocytes. Macroscopic nodules of the hepatocytes on the spleen were seen at about 6 months after transplantation. When the differentiation of the transplanted fetal hepatic tissue was compared with the development of a normal neonatal liver after birth, it was delayed by only about one week, while there was no proliferation of bile ducts in the normal neonatal liver. This experimental model provides a useful system for investigating liver regeneration and the mechanism of cell growth.

Animals↗

PubMed1987