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S Taketani

Publications and source records attributed to S Taketani.

161 records · Page 9Linked to original sources
PubMed1984

Non-enzymatic heme formation in the presence of fatty acids and thiol reductants.

Non-enzymatic heme formation from equimolar amounts of porphyrin and iron was investigated. When mesoporphyrin IX and iron citrate were incubated with oleic acid and dithiothreitol at 37 degrees C in vacuo, mesoheme was formed in a high yield. When protoporphyrin IX and deuteroporphyrin IX were used, protoheme and deuteroheme were formed, respectively. Cysteine or 2-mercaptoethanol instead of dithiothreitol also resulted in the formation of heme. Linoleic acid was as effective as oleic acid, but at 37 degrees C, saturated fatty acids and phospholipids gave low yields. When incubation was at 70 degrees C saturated fatty acids as well as unsaturated fatty acids produced a large amount of heme. The optimum pH was 8.8. By increasing the concentration of Triton X-100 to 0.1%, heme formation decreased, and at concentrations above this level, completely disappeared. The conditions of non-enzymatic heme reaction presented here seem to be useful in elucidation of the mechanism of metalloporphyrin formation.

Fatty Acids↗
PubMed1984

Structural analysis of an HLA-B7 antigen variant detected by cytotoxic T lymphocytes.

It has been demonstrated previously that lymphocytes of donor CF (HLA-A29,w33; B7,14) are not recognized by the HLA-B7-specific CTL clone HG-31. This report presents a structural comparison of the HLA-B7 antigen of donor CF with a "normal" HLA-B7 antigen, derived from the cell line JY. Isoelectric focusing showed that CF HLA-B7 heavy chains were more acidic than JY HLA-B7 heavy chains by the equivalent of a single charge. High pressure liquid chromatography and ion exchange chromatography comparisons of double-labeled tryptic peptides revealed a single detectable difference, which corresponded to the tryptic peptide spanning residues 112 to 121 on the HLA-B7 heavy chain. Although the complete amino acid sequence of this peptide was not obtained, the partial sequence indicates a substitution of an unidentified amino acid for tyrosine at position 116 of the heavy chain. This residue is found to vary among HLA specificities and to be altered in many H-2Kb mutants.

Amino Acid Sequence↗
PubMed1983

HLA-A2 mutants immunoselected in vitro. Definition of residues contributing to an HLA-A2-specific serological determinant.

The HLA-A2-specific mouse monoclonal antibody BB7.2 plus complement has been used to immunoselect variant clones of the lymphoblastoid cell line T5-1 (HLA-A1, -A2, -B8, and -B27). Members of one class of variant clones appear to express cell surface HLA-A2 molecules that display reduced reactivity with the selecting antibody, but normal or near normal reactivities with some other HLA-A2-specific monoclonal antibodies and human alloantisera. The HLA-A2 heavy chains derived from two of these variant clones were characterized by comparative double-label tryptic peptide mapping in conjunction with microsequence analysis. These heavy chains were found to carry distinct mutations in the same peptide in the molecule. We conclude that residues within this short segment of the polypeptide contribute to an HLA-A2-specific serological determinant.

Amino Acid Sequence↗
PubMed1983

Structural analysis of HLA-A2 antigen from immunoselected mutant 8.6.1: further definition of an HLA-A2-specific serological determinant.

The HLA-A2 mutant cell line 8.6.1 was isolated previously from the lymphoblastoid B cell line T5-1 (HLA-A1, -A2, -B8, and -B27) by immunoselection with the mouse HLA-A2-specific monoclonal antibody BB7.2 and complement. The HLA-A2 molecules synthesized by 8.6.1 do not react with either the selecting antibody or with a second HLA-A2-specific monoclonal antibody, PA2.1. In this study, HLA-A2 heavy chains derived from 8.6.1 and those from the parent T5-1 cells have been analyzed by double-labeled tryptic peptide mapping by using reverse-phase HPLC, cation exchange chromatography, and microsequence analysis. We detect only a single difference between these molecules: 8.6.1 HLA-A2 differs from T5-1 HLA-A2 by the substitution of lysine for glutamic acid at position 161. This result is consistent with data derived from other immunoselected mutants, which implicate the second heavy chain domain (alpha 2) in the expression of the PA2.1 and BB7.2 epitopes, and suggests a crucial role for glutamic acid at position 161 in the formation of an HLA-A2-specific determinant.

Amino Acid Sequence↗
PubMed1982

Comparative structural analysis of HLA-A2 antigens distinguishable by cytotoxic T lymphocytes: variants M7 and DR1.

Comparative primary structural analyses have begun to elucidate polymorphic residues and segments of the class I antigens of the major histocompatibility complex, at least some of which presumably contribute to determinants important in immune recognition events. HLA-A2 structural variants have been described which are serologically indistinguishable from other HLA-A2 antigens, yet which can be recognized neither by HLA-A2 specific alloimmune nor by HLA-A2 restricted, virus immune cytotoxic T lymphocytes. This study utilizes double-label tryptic peptide comparisons in combination with both conventional and microsequence analyses to investigate the structure of two such variants, M7 and DR1. We find that these variants are identical with each other and differ from the predominant HLA-A2 heavy chain species by a glutamine to arginine substitution at residue 43, by an unidentified substitution in the tryptic peptide spanning residues 147-157, and by an as yet poorly defined alteration in glycosylation. Structural information from these and other variants should be useful in precisely defining functionally important determinants on the molecule.

Amino Acid Sequence↗
PubMed1982

Purification and substrate specificity of bovine liver-ferrochelatase.

Bovine ferrochelatase from liver mitochondria was purified 1434-fold with a 31% yield to apparent homogeneity by a procedure involving solubilization, ammonium sulfate fractionation and blue Sepharose CL-6B chromatography. The molecular weight of the homogeneous protein was 42 500 when measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 200 000 was obtained by Sepharose 6B gel filtration. The specific activity for mesoheme synthesis was 413 nmol x mg protein-1 x min-1 at 37 degrees C and for protoheme synthesis 88 nmol x mg-1 x min-1. The optimum pH was 8.0 and Km values for the substrates were: protoporphyrin IX, 54 microM; mesoporphyrin IX, 46 microM; iron with protoporphyrin IX, 46 microM, iron with mesoporphyrin IX, 44 microM. The purified enzyme inserted iron into the following dicarboxylic porphyrins in descending order: meso-, deutero-, 2,4-diacetyldeutero-, hemato-, and protoporphyrin IX. This did not take place in the case of 2,4-diformyldeuteroporphyrin IX. Porphyrin c was converted to only a negligible amount of heme c, and coproporphyrin III did not act as a substrate at all. When metal specificity was examined, the highest value was obtained with zinc, decreasing in order with iron, cobalt and nickel. The enzyme failed to catalyze the insertion of copper or manganese into porphyrin. An antibody specific for the purified bovine ferrochelatase was prepared, and studies confirmed that the synthetic activities of iron-porphyrin, zinc-porphyrin and cobalt-porphyrin are ascribable to ferrochelatase.

Animals↗
PubMed1981

Rat liver ferrochelatase. Purification, properties, and stimulation by fatty acids.

Ferrochelatase from rat liver mitochondria was purified 628-fold with a 25% yield to apparent homogeneity. The purification procedure involved solubilization of the enzyme with sodium cholate, followed by ammonium sulfate fractionation and blue Sepharose CL-6B column chromatography. The molecular weight of the enzyme was estimated to be 42,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Sepharose 6B gel filtration gave a molecular weight of 240,000. The purified enzyme was analyzed for amino acid composition and shown to have abundant amounts of lysine (11%) and hydrophobic amino acid residues (48%). The enzyme was quite stable in a buffer containing 20% glycerol and 1 mM dithiothreitol. When the enzyme was assayed in the presence of palmitic acid, the specific activity for mesoheme synthesis was approximately 12,000 nmol formed/30 min/mg of protein at 37 degrees C; for protoheme synthesis, a value of 3,500 nmol formed/30 min/mg of protein was obtained. The optimum pH for the reaction was 7.8, and the Km values for the substrates were as follows: protoporphyrin IX, 28.5 microM; mesoporphyrin IX, 26.7 microM; iron with protoporphyrin IX, 33.1 microM; and iron with mesoporphyrin IX, 37.4 microM. Enzyme activity was inhibited by metals such as Co, Zn, Pb, Cu, or Mn and was highly sensitive to sulfhydryl inhibitors. The purified enzyme contained fatty acids, and its activity was markedly stimulated by their addition. Phospholipids slightly stimulated enzyme activity. Short chain carbonic acids and neutral lipids produced no effects.

Amino Acids↗
PubMed1981

Subcellular localization of the enzymes involved in the late stage of ergosterol biosynthesis in yeast.

The subcellular distribution of enzymes involved in the reaction sequence from zymosterol to ergosterol was studied. The spheroplasts obtained from cells aerobically grown on ethanol were gently disrupted and the homogenate was fractionated into the subcellular organelles by differential centrifugation. Inspection of the distribution of several marker enzymes revealed that the fractionation was reasonably effected. As a result of experiments, delta 8-delta 7-sterol isomerase, S-adenosylmethionine: delta 24-sterol methyltransferase and the enzyme involved in the reaction sequence from episterol to ergosterol were localized in microsomes. These results suggest the localization of enzymes involved in the late stage of ergosterol synthesis in microsomes.

Cholestadienols↗
PubMed1981

Purification and properties of sterol-ester hydrolase from Saccharomyces cerevisiae.

Sterol-ester hydrolase [EC 3.1.1.13] from Saccharomyces cerevisiae grown aerobically was solubilized with 1% Tween 20 and purified about 700-fold by the protamine sulfate treatment, DEAE-cellulose-, Sepharose 6B- and DEAE-cellulose column chromatographies. The molecular weight of the enzyme was estimated to be 70,000 by Sepharose 6B gel filtration. The enzyme activity showed two peaks of pH optimum at 4.4 and 6.8. Triton X-100 stimulated the activity as its low concentrations at both pH regions, but decreased the activity at its high concentrations at pH 6.8. The presence of Tween 20 or Tween 80 also stimulated the activity. These results were different from those in the previous report showing no stimulation of the crude enzyme by these detergents. The stimulation of the activity by phosphatidylcholine or low concentrations of lysophosphatidylcholine was similar to that by Triton X-100, and taurocholate was less effective than Triton X-100. The enzyme activity was inhibited by divalent cations such as Hg2+ and Cu2+.

Carboxylic Ester Hydrolases↗
PubMed1979

Characterization of sterol-ester synthetase in Saccharomyces cerevisiae.

Cell-free extracts of Saccharomyces cerevisiae grown under aerobic as well as semi-anaerobic conditions were found to catalyze the synthesis of fatty acid ester of sterol from cholesterol, fatty acid, ATP and CoA, or from cholesterol and fatty acyl-CoA. This result indicates that the enzyme involved in the formation of the ester is acyl-CoA:sterol O-acyltransferase (EC 2.3.1.26). The enzyme had a broad substrate specificity for sterols and acyl-CoAs. The enzyme levels in the cells grown under aerobic and semi-anaerobic conditions were almost equal. The enzyme was located in the microsomal fraction of the aerobically grown cells.

Acyltransferases↗
PubMed1978

Characterization of sterol-ester hydrolase in Saccharomyces cerevisiae.

A homgenate of Saccharomyces cerevisiae grown under semi-anaerobic as well as aerobic conditions was found to catalyze the hydrolysis of fatty acid esters of sterols in the presence of Triton X-100. The enzyme levels in cells grown under various conditions were similar and the enzyme had a broad substrate specificity for sterol esters. The enzyme was localized in the mitochondrial fraction for the aerobically grown cells and in the mitochondrial and cytosolic fractions for the semi-anaerobically grown cells.

Carboxylic Ester Hydrolases↗
PubMed1978

Quantitative aspects of free and esterified sterols in Saccharomyces cerevisiae under various conditions.

For extraction of free and esterified sterols from yeast cells, a method was devised in which both forms of sterols were extracted with light petroleum after the treatment of the cells with acetone, and then with dimethylsulfoxide. The content of sterol esters in the cells under aerobic conditions markedly increased with time, amounting to 95% of the total sterols under some conditions. However, the formed sterol esters were decreased, accompanied with an increase of free sterols, when the cells were put under anaerobic conditions. Variations of radioactivities of both sterols which had been labeled in the side chain by incubation of the cells with [Me[-14C]methionine were examined on the cells grown under various conditions. No variation was observed on the cells under aerobic conditions. On the other hand, the labeled esters were hydrolyzed to yield free sterols in the cells under anaerobic conditions. In the cells under aerobic conditions, the free sterols were found to consist mainly of ergosterol, whereas the esterified sterols contained considerable amounts of zymosterol, lanosterol, and other intermediate sterols besides ergosterol.

Aerobiosis↗
PubMed1978

Ergosterol biosynthesis in yeast. Pathways in the late stages and their variation under various conditions.

[Methyl-14C]methionine was supplied to yeast cells under aerobic and anaerobic conditions for the investigation of the pathway for ergosterol biosynthesis after the methylation of the side-chain. Under aerobic conditions, the incorporation of radioactivity into ergosterol was high. With a limited oxygen supply, in contrast, the radioactivity was first accumulated in ergosta-7,24(28)-dien-3beta-ol and ergosta-8,24(28)-dien-3beta-ol, and then transferred to ergost-7-en-3beta-ol, ergost-8-en-3beta-ol and ergosta-7,22-dien-3beta-ol with time. Under strictly anaerobic conditions, a double bond was introduced neither to delta5 nor to delta22. The results of the tracer experiments suggested the operation of several pathways in the late stages of ergosterol biosynthesis. It was also suggested that the main pathways varied depending on the conditions such as oxygen supply and other factors. The above conclusion was supported by the results of the analyses of the sterol compositions of the cells grown under various conditions.

Aerobiosis↗
PubMedDate not supplied

Lead and the terminal mitochondrial enzymes of haem biosynthesis.

Lead exposure causes increases in urinary coproporphyrin excretion and the accumulation of zinc protoporphyrin in red cells. In the conventional view of the effect of lead on haem biosynthesis, the accumulation of these metabolites results from lead inhibition of two of the mitochondrial enzymes of haem biosynthesis, coproporphyrinogen oxidase (EC 1.3.3.3) and ferrochelatase (EC 4.99.1.1). This review critically assesses the evidence for the inhibition of these enzymes. We consider this evidence to be inconclusive and alternative explanations for the increased concentrations of coproporphyrin and zinc protoporphyrin are proposed.

Animals↗
PubMedDate not supplied

Nitric oxide gas infusion to the oxygenator enhances the biocompatibility of heparin coated extracorporeal bypass circuits.

Heparin coated bypass circuits have been reported to improve the biocompatibility of extracorporeal circulation, although it is still insufficient and improvable. Nitric oxide (NO) is known to inhibit platelet activation and inflammatory reactions. In this study, the authors evaluated exogenous NO infusion in enhancing the effect of a heparin coated bypass circuit on the biocompatibility of an extracorporeal circuit, especially in view of the attenuation of the inflammatory response. A miniature closed bypass circuit, including an oxygenator (BioActive surface; Carmeda, Stockholm, Sweden) was primed with fresh human heparinized blood and perfused with a centrifugal pump. Either pure N2 gas (control group: n = 7) or NO gas (NO group [100 ppm in N2]: n = 7) was infused to the oxygenator. NO metabolites (nitrite and nitrate), platelet count, thrombin-antithrombin III complex (TAT), alpha2-plasmin-plasminogen inhibitor complex (PIC), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4), serotonin, complement 3 activation products (C3a), granulocyte elastase, and bradykinin were measured at 0, 30, 60, 120, and 180 min after starting perfusion. At every sampling point, platelet counts were significantly higher, and TAT, beta-TG, and bradykinin were lower in the NO group than in the control group. PF4, C3a, and granulocyte elastase were significantly lower in the NO group at 60, 120, and 180 min. These results suggest that NO gas infusion to the oxygenator enhances the biocompatibility of heparin coated extracorporeal circuits.

Biocompatible Materials↗
PubMedDate not supplied

Proteasome inhibitor 1 enhances paclitaxel-induced apoptosis in human lung adenocarcinoma cell line.

Paclitaxel is a chemotherapeutic drug that induces apoptosis in tumor cells by stabilizing microtubules, prevents normal mitosis, and blocks the cell cycle at the G2/M phase. We have previously reported that the activation of caspase-3 and caspase-8 plays a crucial role in paclitaxel-induced apoptosis. Anti-tumor reagents including paclitaxel, irradiation, and other stimuli activate the transcription factor NF-kappaB, which has the ability to suppress the apoptotic potential of those stimuli. Using a human lung adenocarcinoma cell line (LC-2-AD), we therefore examined whether the inhibition of NF-kappaB activity by proteasome inhibitor 1 (PS1) could become a new adjuvant therapy for cancer. A synergistic effect on apoptosis induction was observed with the combination of more than 0.1 microg/ml paclitaxel and 0.5 microM PS1. An increase in the cell number of apoptotic cells is correlated with the loss of Deltaphim and the activation of caspase-3 and caspase-8. Furthermore, augmented apoptosis is related to NF-kappaB activation. Based on these findings, we propose that the combination of paclitaxel with PS1 could be a new strategy for cancer treatment.

Adenocarcinoma↗