Biomedical subjects

Y Banno

Publications and source records attributed to Y Banno.

At least 109 records · Page 6Linked to original sources

PubMed1987

Stimulation by GTP-binding proteins (Gi, Go) of partially purified phospholipase C activity from human platelet membranes.

A phospholipase C exhibiting preferential hydrolytic activity for polyphosphoinositides was partially purified from the deoxycholate extract of human platelet membranes by Q-Sepharose and Heparin-Sepharose column chromatographies. The activity of this purified phospholipase C free of the GTP gamma S-binding activity was stimulated at a similar level by addition of purified rat brain Gi or Go. These results suggest that GTP-binding proteins may interact directly with a solubilized membrane phospholipase C to stimulate its activity.

Animals↗

PubMed1987

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis.

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).

Acid Phosphatase↗

PubMed1987

Inhibitory effect of pentobarbital on phospholipase C activity in ischaemic rat brain.

Ischaemic rat brains were examined for temporal changes in phospholipase C activity with phosphatidylinositol; the effects of pentobarbital on the activity also were investigated. Ischaemia was produced by decapitation. Pentobarbital (60 mg/kg) was administered i.p. for 15 min before decapitation. The removed heads were incubated at 37 degrees C for 1, 5, 15 or 30 min and then quickly frozen in liquid nitrogen. After isolation of subcellular fractions from the brains, phospholipase C activity was measured for cytosol and microsomes, using radioactive phosphatidylinositol as a substrate. The results demonstrated that brain phospholipase C predominantly localized in the cytosol was dependent on Ca2+ for full activity and had neutral pH optima. Although the enzyme activity did not increase during ischaemia, pentobarbital inhibited phospholipase C activity in the cytosol but not in the microsomes. These observations suggest that pentobarbital may exert a cerebral protective action due to, at least in part, the repression of phospholipase C followed by a reduction of phosphatidylinositol breakdown, preventing perturbation to the integrity of membranes, during ischaemia.

Animals↗

PubMed1986

GTP and GDP will stimulate platelet cytosolic phospholipase C independently of Ca2+.

The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C.

Blood Platelets↗

PubMed1986

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms.

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.

Blood Platelets↗

PubMed1986

Properties of lysosomal beta-hexosaminidase accumulated in Niemann-Pick mouse liver.

Various lysosomal acid hydrolases from tissues of Niemann-Pick mice, a mutant strain of C57BL/KsJ mice (spm/spm), were examined and compared to those from control mice. Activities of beta-hexosaminidase, beta-galactosidase, acid phosphatase, and cathepsin L were elevated in the liver and spleen of the affected mice, whereas no significant changes in beta-glucosidase and acid alpha-glucosidase were observed. Alpha-Mannosidase and neutral alpha-glucosidase activities were rather decreased in the affected mouse liver. The level of beta-hexosaminidase in the Niemann-Pick mice was raised sixfold in the liver and two- to threefold in the spleen and brain, whereas its total activity was decreased in the kidney. Sixty to ninety percent of total activity of lysosomal hydrolases was solubilized with 0.1% Triton X-100 in control mice, but most of the beta-hexosaminidase activity of the Niemann-Pick mice remained associated with the membrane fraction of liver lysosomes. The beta-hexosaminidase of the Niemann-Pick mice was appreciably stable when heated at 55 degrees C, while hydrolases of the affected mice and all of the enzymes tested in control mice were heat labile. The relative content of two beta-hexosaminidase fractions separated by DEAE-cellulose column chromatography was 8% for beta-hexosaminidase I and 92% for beta-hexosaminidase II in the case of the control mouse liver. The isozyme pattern of hexosaminidases in Niemann-Pick mice was similar to that of control enzymes. However, the beta-hexosaminidase II accumulated in Niemann-Pick mouse liver was different from that of the control in optimum pH, Km values and thermostability.

Animals↗

PubMed1986

Properties of acid phospholipases in lysosome and extracellular medium of Tetrahymena pyriformis.

Phospholipase activity in the lysosomes of the protozoan Tetrahymena pyriformis strain NT-1 was studied using phospholipids radioactively labeled in the fatty acid moieties. Lysosomal homogenates showed high phospholipase activity with an acidic pH optimum. Unlike the phospholipases in rat liver lysosomes, almost all activity was recovered from the membranous fraction of the lysosomes. The activity was partially solubilized by treatment of the membranes with a detergent or trypsin. Using specifically labeled phospholipids revealed that phospholipase. A1 and C are predominant in Tetrahymena lysosomes, no appreciable phospholipase A2 or lysophospholipase activity was detected in the fraction. There are two catabolic pathways of the hydrolysis of phospholipid: Hydrolysis is initiated by deacylation at the 1-position by phospholipase A1 and the 2-acyllysophospholipid thus formed is successively attacked by (lyso)phospholipase C; hydrolysis is initiated by cleavage of phosphodiester by phospholipase C and the diacylglycerol thus formed is attacked by lipase. Both pathways give the same end products, free fatty acid and 2-monoacylglycerol. The former pathway might be predominant in Tetrahymena lysosomes under physiological conditions since the pathway is independent of detergent. Phospholipases A1 and C activities were partially released into the medium. At least two different phospholipases C are present in the medium as judged by chromatographic behavior and their substrate specificities.

Animals↗

PubMed1986

Involvement of the membrane fluidity of lactosylceramide-targeted liposomes in their intrahepatic uptake.

Incorporation of N-lignoceroyldihydrolactocerebroside (lactosylceramide) enhanced liver uptake of small unilamellar liposomes consisting of dipalmitoylphosphatidylcholine, cholesterol and dicetyl phosphate (molar ratio, 4:5:1). The increase in liver uptake was mostly accounted for by an enhanced uptake into the parenchymal cells. The enhancing effects of lactosylceramide on uptake of the liposomes into liver in vivo and into isolated parenchymal cells in vitro were greater with dipalmitoylphosphatidylcholine-based liposomes than with dimyristoylphosphatidylcholine-based ones. In contrast, addition of lactosylceramide had no significant effect on egg phosphatidylcholine vesicle uptake. The stimulated uptake of lactosylceramide liposomes by parenchymal cells was counteracted by added asialofetuin. These observations suggest that transfer of the targeted liposomes via a galactose-specific receptor into parenchymal cells may be controlled by the membrane fluidity of the liposomes.

Animals↗

PubMed1985

Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis.

The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.

Acid Phosphatase↗

PubMed1985

Secreted phospholipases of the dimorphic fungus, Candida albicans; separation of three enzymes and some biological properties.

Several phospholipases are secreted into the culture medium by growing yeast cells of Candida albicans 3125. DEAE-Sephadex column chromatography of concentrated culture filtrate revealed three separable fractions with phospholipase activities. Analysis of products of hydrolysis showed that the enzyme activities were lysophospholipase, lysophospholipase-transacylase and a phospholipase B.

Acyltransferases↗

PubMed1985

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena.

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.

Amino Acids↗

PubMed1985

Intracellular phospholipase activities of Tetrahymena pyriformis.

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.

Animals↗

PubMed1984

A mouse model for Niemann-Pick disease: phospholipid class and fatty acid composition of various tissues.

Recently, a strain of mice bearing an autosomal recessive gene, spm, has been described. On the basis of clinical and pathological findings these mice have been suggested as a useful model of human Niemann-Pick disease. Phospholipids and their fatty acid compositions were analyzed for liver, spleen, whole brain, erythrocytes, and blood plasma from "Niemann-Pick"animals (spm/spm) and heterozygous controls (spm/+). Sphingomyelin and bis(monoacylglycero)phosphate accumulated in the liver and spleen of the affected mice, whereas no significant proportional change of phospholipids was observed in the whole brain. The phospholipid composition in erythrocytes and blood plasma of the homozygous mice was not different from that of the heterozygous controls. The fatty acyl chain profile of accumulated bis(monoacylglycero)phosphate was characterized by the high content (more than 80%) of unsaturated fatty acids; the main components were oleic acid, linoleic acid, and docosahexaenoic acid. A high unsaturation index of the fatty acyl chain was found in sphingomyelin accumulated in organs and in almost all phospholipids of brain, erythrocytes, and blood plasma of "Niemann-Pick" mice. It is conceivable that desaturation of fatty acids is enhanced in the "Niemann-Pick" mice.

Animals↗

PubMed1983

Purification and characterization of a secreted protease from Tetrahymena pyriformis.

A simple major protease, secreted into the medium during growth of Tetrahymena pyriformis strain W, has been purified about 4000-fold by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and affinity chromatography on organomercurial-Sepharose. The purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22 000-23 000. Amino acid analysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena protease consists of multiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5-8.0 with alpha-N-benzoyl-DL-arginine-p-nitroanilide and with azocasein, while it was in the lower pH range (4.5-5.5) for denatured hemoglobins. The purified enzyme was inhibited by compounds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.

Amino Acids↗

PubMed1983

Targeting of asialofetuin sugar chain-bearing liposomes to liver lysosomes.

Specific direction of liposomes bearing an asialofetuin sugar chain (AFSC) to liver parenchymal cells was examined both in vivo and in vitro. The AFSC-bearing liposomes were preferentially recovered in the liver within several minutes after an intravenous injection into mice and were found to be predominantly localized in mitochondrial-lysosomal fraction. The massive distribution of the AFSC-liposomes in this fraction was also confirmed by using a lysosomal protease inhibitor, E-64-d. In isolated rat hepatocytes, the uptake of AFSC-liposomes was increased 2-3-fold as compared with the control liposomes without AFSC. Thus liposomes bearing AFSC would be useful to target enzymes to liver lysosomes.

Animals↗

PubMed1982

Changes in particulate-bound protease activity during cold acclimation in Tetrahymena pyriformis.

The protease activity, as assayed at pH 8.0 with azocasein as substrate, of a ciliate protozoan Tetrahymena pyriformis NT-1, was found to alter by growing the cells at various constant temperatures or at shifted temperatures. The intracellular protease activity, when cells were grown at either constant 39 degrees C or 15 degrees C, was decreased throughout the growth phase with significant secretion into the medium. On the other hand, when the culture temperature was transferred from 39 degrees C to 15 degrees C, the protease activity in cells was greatly increased up to about 28-fold at 8 h after the shift. There was, however, no secretion into the medium during the cold acclimation after the shift, where no cell division occurred. The elevated protease activity was quickly decreased to the control level when the culture was warmed to 39 degrees C after 8-h chilling, and recovery of normal cell division was seen. The marked increase in the protease activity caused by the shift to 15 degrees C was completely blocked by the addition of either cycloheximide or actinomycin D. The thermally induced enhancement of protease activity was found to occur with no different preference between three protease fractions.

Adaptation, Physiological↗

PubMed1982

Studies on thermal adaptation in Tetrahymena membrane lipids. Modification of positional distribution of phospholipid acyl chains in plasma membranes, mitochondria and microsomes.

The positional distribution of fatty acyl chains in major glycerophospholipids (phosphatidylethanolamine, phosphatidylcholine and 2-aminoethylphosphonolipid) of three membrane fractions (plasma membranes, mitochondria and microsomes) of the thermotolerant Tetrahymena pyriformis NT-1 cells was analyzed at various time intervals within 10 h after a temperature shift from 39 to 15 degrees C. During this period of acclimation there were no changes in both the total phospholipid content and its proportional composition. At the 1-position, the content of palmitate in phosphatidylethanolamine (present solely in diacyl type), diacylphosphatidylcholine, and diacyl-(2-aminoethyl)phosphonolipid was decreased progressively after temperature-shift, while gamma-linolenate increased in a complementary fashion, in mitochondria and microsomes. The increase in the percentage of linoleate was compensated by the decrease in oleate at the 2-position of two 1,2-diacylphospholipids. On the other hand, as for 1-alkyl-2-acyl-phospholipids, a marked increment in gamma-linolenate occurred, with a decline of oleate and linoleate at the 2-position of 1-alkyl-2-acyl-phosphatidylcholine, but no significant alterations were seen at the 2-position of 1-alkyl-2-acyl-(2-aminoethyl)phosphonolipid. The results suggest that the newly modified phospholipid molecular species such as 1-gamma-linolenoyl-2-linoleoyl-phosphatidylethanolamine and 1-hexadecyl-2-gamma-linolenoyl-phosphatidylcholine disseminate rapidly to other cell compartments and that they would play a pivotal role in the adaptive amelioration of altered membrane physical states during the cold acclimation.

Animals↗

PubMed1982

Biochemical characterization of secreted proteases during growth in Tetrahymena pyriformis WH-14: comparison of extracellular with intracellular proteases.

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and alpha-N-benzoyl-DL-arginine-rho-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000-24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3 and 1.4 mM for P-I, P-II, P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S-1 . M-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.

Amino Acids↗