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Biomedical subjects

R Taguchi

Publications and source records attributed to R Taguchi.

At least 73 records · Page 4Linked to original sources
PubMed1985

Ectoenzyme release from rat liver and kidney by phosphatidylinositol-specific phospholipase C.

Ectoenzyme release from rat liver and kidney by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphatase and 5'-nucleotidase were released from rat kidney slices to extents of up to 60% and 30%, respectively. Release of alkaline phosphatase was observed at lower amounts of PI-specific phospholipase C than that of 5'-nucleotidase. Both enzymes were more easily released from microsomal fractions or free cells. From kidney cells, alkaline phosphatase was released without cell lysis, and more than 80% release of alkaline phosphatase was observed at 3.8% hydrolysis of PI. Isoelectric focusing profiles of alkaline phosphatase released by PI-specific phospholipase C were significantly different from the control in the cases of both rat liver and kidney. Lubrol-solubilized alkaline phosphatase was eluted at the void volume of a Toyopearl HW-55 column, while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the lower-molecular-weight region corresponding to 100,000-110,000 daltons. Furthermore, Lubrol-solubilized phosphatase became more thermostable on treatment with PI-specific phospholipase C.

5'-Nucleotidase↗
PubMed1984

Acetylcholinesterase release from mammalian erythrocytes by phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis and characterization of the released enzyme.

The mode of acetylcholinesterase release from mammalian erythrocyte membranes by the action of phosphatidylinositol(PI)-specific phospholipase C of Bacillus thuringiensis was studied. As regards intact erythrocytes, a larger amount of acetylcholinesterase was released from sheep or bovine erythrocytes than from horse erythrocytes. From horse erythrocyte ghosts, acetylcholinesterase was more easily released than from intact cells. Bovine erythrocyte acetylcholinesterase released by PI-specific phospholipase C was purified by column chromatography on DEAE-cellulose, affinity gel and Sepharose 6B, to a homogeneous state, as indicated by polyacrylamide gel electrophoresis, with a recovery of 39%. Also, bovine erythrocyte acetylcholinesterase was solubilized by Triton X-100 and partially purified. The properties of these acetylcholinesterase preparations obtained by the action of PI-specific phospholipase C and/or Triton X-100 were studied in detail. On elution from the Sepharose 6B column, Triton X-100-solubilized acetylcholinesterase was eluted at the void volume while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the region corresponding to M.W. 250,000. Furthermore, the heat stability of acetylcholinesterase purified after solubilization with PI-specific phospholipase C was higher than that of the Triton X-100-solubilized acetylcholinesterase. The close association and direct interaction of PI with acetylcholinesterase in the erythrocyte membrane was suggested by the above results.

Acetylcholinesterase↗
PubMed1984

Phosphatidylinositol-hydrolyzing enzymes in chicken liver cells.

Phosphatidylinositol-hydrolyzing activities were found in mitochondrial, lysosomal, microsomal, and cytosol fractions of chicken liver. At least two different activities were detected; cytosolic activiti(es) was maximally exhibited around pH 6.0, activated by Ca2+, and inhibited by EDTA; whereas lysosomal activiti(es) had a optimal pH 5.0, being unaffected by Ca2+ or EDTA.

Animals↗
PubMed1983

Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes.

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.

Adsorption↗
PubMed1983

Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C.

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.

Bacillus cereus↗
PubMed1983

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on the glucose-6-phosphatase system of rat liver microsomes.

The role of phospholipids in the glucose-6-phosphatase system, including glucose-6-P phosphohydrolase and glucose-6-P translocase, was studied in rat liver microsomes by using phospholipases C and detergents. In the time course experiments on detergent exposure, the maximal activation of glucose-6-P phosphohydrolase varied according to the nature of the detergent used. On treatment of microsomes with phospholipase C of C. perfringens, the activity of glucose-6-P phosphohydrolase without detergent (i.e. without rupture of translocase activity) was gradually decreased with the progressive hydrolysis of phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane, and was restored by incubation of these microsomes with egg yolk phospholipids. The extent of decrease in this phosphohydrolase activity in the detergent-exposed microsomes (with rupture of translocase activity) also varied depending on the detergent used (Triton X-114 or taurocholate). When 66% of the phosphatidylinositol on the membrane was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the inhibition of glucose-6-P phosphohydrolase activity without detergent was very small. Although the inhibition of enzyme activity with detergent was apparently greater than that without detergent, the enzyme activity was stimulated by the breakdown of phosphatidylinositol when the enzyme activity was measured at lower concentration (0.5 mM) of substrate, glucose-6-P. The latency of mannose-6-P phosphohydrolase, a plausible index of microsomal integrity, remained above 70% after the hydrolysis of phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. The results show that the glucose-6-phosphatase system requires microsomal phospholipids for its integrity, suggesting that there exists a close relation between phosphatidylinositol and glucose-6-P translocase.

Animals↗
PubMed1983

Studies on the interactions between phospholipids and membrane-bound enzymes in microsomes. Effects of phospholipases C on kinetic properties of the glucose-6-phosphatase system in rat liver microsomes.

Through kinetic analysis, the relationships between the glucose-6-phosphatase system and constituent phospholipids were studied in rat liver microsomes. When phosphoglycerides such as phosphatidylcholine and phosphatidylethanolamine on the microsomal membrane were hydrolyzed by phospholipase C of C. perfringens, the activities of glucose-6-P phosphohydrolase and glucose-6-P:glucose phosphotransferase both decreased with or without subsequent exposure to taurocholate. In these cases, the Michaelis constants (Km) for glucose-6-P were increased, concomitant with the decrease in the maximal velocities (Vmax) for glucose-6-P hydrolysis. On exposure to taurocholate, the apparent Km for glucose of phosphotransferase was decreased. When phosphatidylinositol was hydrolyzed by phosphatidylinositol-specific phospholipase C of B. thuringiensis, the activities of phosphohydrolase and phosphotransferase were both decreased on exposure to taurocholate. In this case, the value of Vmax of phosphohydrolase was decreased and that of Km for glucose-6-P was slightly decreased, while the apparent Km for glucose of phosphotransferase was increased. Without exposure to detergent, the activities of phosphohydrolase and phosphotransferase both decreased at glucose-6-P concentrations higher than 10 mM. However, at a concentration lower than 1 mM, the activity of phosphohydrolase became higher than that of the control, and Vmax and Km for glucose-6-P were decreased. A similar tendency was also observed in microsomes where membranous phosphatidylinositol was hydrolyzed, when they were treated with DIDS (an anion-transport inhibitor). From these results, it is concluded that the activity of glucose-6-phosphatase is greatly influenced by changes of the phospholipids on the microsomal membrane, and the activity of glucose-6-P translocase is stimulated by the breakdown of phosphatidylinositol.

Animals↗
PubMed1983

The action of sphingomyelinase of Bacillus cereus on bovine erythrocyte membrane and liposomes. Specific adsorption onto these membranes.

Sphingomyelinase of Bacillus cereus was specifically adsorbed onto sphingomyelin liposome in the presence of Ca2+ or with the coexistence of Ca2+ and Mg2+, but not onto the liposome of phosphatidylcholine. In the presence of Ca2+, the enzyme adsorption onto bovine erythrocytes and liposome increased with an increase in the amount of sphingomyelin. These results support that the major binding site for sphingomyelinase in the erythrocytes is sphingomyelin. The temperature-dependence of enzyme adsorption was not influenced by a change in ATP content of bovine erythrocytes. After treatment of red cells with neuraminidase or pronase, enzyme adsorption at 0 degrees C lower than that at 37 degrees C was observed. In unsealed or right-side-out ghosts, the difference between the enzyme adsorption at 37 degrees C and that at 0 degrees C became less pronounced than in the erythrocytes. Furthermore, the extent of enzyme adsorption onto sphingomyelin liposome at 0 degrees C was almost equal to that at 37 degrees C. The enzyme adsorption onto the erythrocyte membrane and liposome was always enhanced in the presence of Ca2+; in the presence of Mg2+ alone, adsorption was observed only for erythrocyte ghosts and the adsorbed enzyme was released from the membrane after extensive degradation of sphingomyelin by sphingomyelinase. With the coexistence of Ca2+ and Mg2+, the enzymatic hydrolysis of sphingomyelin proceeded rapidly for the attack against liposome and all the membranes tested, whereas in the presence of Mg2+ alone, hydrolysis was observed only for the action of liposome and ghosts. No appreciable hydrolysis of sphingomyelin was observed in the presence of Ca2+ nor in the absence of divalent metal ions.

Adenosine Triphosphate↗
PubMed1983

Complete purification of phosphatidylinositol-specific phospholipase C from a strain of Bacillus thuringiensis.

A phosphatidylinositol-specific phospholipase C was purified from the culture broth of Bacillus thuringiensis IAM 12077 to a homogeneous state as revealed by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 559 units/mg and recovery of the enzyme activity was 31%. Molecular and physiological properties of the purified enzyme, including molecular weight (22,000), isoelectric point (pI = 4.9) and its ectoenzyme-releasing activity, were studied in comparison with those other known enzymes of bacterial origin.

Bacillus thuringiensis↗
PubMed1982

Molecular properties and kinetic studies on sphingomyelinase of Bacillus cereus.

A sphingomyelinase of Bacillus cereus was purified to a homogeneous state (512 U/mg, 2200-fold) as indicated by SDS-polyacrylamide gel electrophoresis and the molecular weight (23,300) was determined by sedimentation equilibrium. The enzyme contained loosely-bound magnesium atom. The addition of Mg2+ accelerated the enzyme reaction regardless of substrates and their physical state. The addition of Ca2+ also accelerated the enzyme reaction slightly, when water-soluble substrates, i.e., 2-hexadecanoylamino-4-nitrophenylphosphorylcholine and p-nitrophenylphosphorylcholine, were used as substrates. On the other hand, the addition of Ca2+ inhibited enzyme reaction when mixed micelles of either sphingomyelin and Triton X-100 or sodium deoxycholate were used. The surface charge on mixed micelles affected the enzyme reaction. When the mixed micelle of sphingomyelin and Triton X-100 was used as substrate, Ca2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 33 microM. On the other hand, when the mixed micelle of sphingomyelin and sodium deoxycholate was used as substrate, Ca2+ stimulated the enzyme reaction at lower concentration in the presence of a low concentration of Mg2+, although higher concentrations of Ca2+ were still inhibitory. In this case, added Ca2+ may be used as a substitute of Mg2+ to neutralize the negative charge on the mixed micelle, improving the accessibility of sphingomyelinase to the micellar substrate. A cationic detergent, cetyltrimethylammonium bromide, seemed to denature or inactivate the enzyme.

Bacillus cereus↗
PubMed1980

Purification and properties of phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis.

A phosphatidylinositol-specific phospholipase C was purified from the culture broth of Bacillus thuringiensis to a homogeneous state as indicated by polyacrylamide gel electrophoresis. Specific activity of purified enzyme was 312 units/mg, and the recovery of the enzyme activity was 27.2%. The purified enzyme (molecular weight: 23 000 +/- 1000) was maximally active at pH 7.5 and not influenced by EDTA. The enzyme specifically hydrolyzed phosphatidylinositol, but did not act on phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and sphingomyelin. The products from phosphatidylinositol of enzyme reaction were diacylglycerol and myoinositol 1,2-cyclic phosphate. The enzyme activity was stimulated by sodium deoxycholate or Triton X-100. Divalent cations such as Ca2+, Mg2+ and Zn2+ were inhibitory at concentrations above 10(-3) M. KCl and NaCl were inhibitory at the concentration higher than 10(-2) M. Alkaline phosphatase, an ecto-enzyme located on the surface of plasma membrane, was released from the slices of rat liver, kidney, pancreas and intestine by the treatment with this phospholipase.

Alkaline Phosphatase↗
PubMed1979

Capsimycin, a new antibiotic. I. Production, isolation and properties.

Capsimycin is a new antifungal antibiotic produced by a strain of Streptomyces sp. C 49--87. The active substance in the fermented broth was isolated by solvent extraction followed by silica gel column chromatography. The antibiotic melts at 186 degrees C (decomp.) and has a molecular formula C30H40N2O6. It exhibits marked inhibitory activity against Phytophthora capsici (Leaf blight disease of cucumber) and Pythium debaryanum (Damping-off disease of cucumber).

Animals↗
PubMed1978

Studies on sphingomyelinase of Bacillus cereus. I. Purification and properties.

A sphingomyelinase was purified 980-fold with recovery of 25.6% from the culture broth of Bacillus cereus, by (NH4)2SO4 precipitation and chromatography on CM-Sephadex, DEAE-cellulose and Sephadex G-75. The purified preparation was free of lipase, protease and other phospholipases. The enzyme specifically hydrolyzed sphingomyelin to ceramide and phosphorylcholine. Lysophosphatidylcholine was also attacked by the enzyme. The enzyme (Mr = 24 000) was maximally active at pH 6-7. Other properties of the enzyme, including hemolytic activity and activation/inhibition studies, are reported.

Bacillus cereus↗