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1.
Properties of Particulate and Detergent-Solubilized Phospholipid N-Methyltransferase Activity from Calf Brain 总被引:4,自引:2,他引:2
Abstract: Calf brain membranes catalyze the enzymatic transfer of [CH3-3H]methyl groups from S-adenosyl-l -[CH3-3H]methionine into endogenous phosphatidyl-N-methylethanolamine (PME), phosphatidyl-N,N-dimethylethanolamine (PDE), and phosphatidylcholine (PC). Phospholipid N-methylation can be stimulated by the addition of exogenous PME or PDE, added in aqueous dispersions with sodium taurocholate. When membranes are incubated in the presence of exogenous PME, [CH3-3H]PDE represents 86% of the labeled phospholipid products. When exogenous PME is replaced by PDE, 91% of the label is incorporated into PC. Thus, under these in vitro conditions it is possible to assay PME- and PDE-N-methyitransferase activity separately. The calf brain phospholipid N-methyltransferase activity has also been solubilized by treating the membranes ultrasonically in the presence of Triton X-100 and 10 mM monothioglycerol. When the detergent extracts are incubated in the presence of exogenous PME, [CH3-3H]PDE represents 86% of the enzymatically labeled products. In the presence of exogenous PDE, more than 97% of the label is incorporated into PC. Optimal conditions for the membrane-bound and detergent-solubilized PME- and PDE-N-methyltransferase activity have been established. These conditions have been used as a basis for testing the hypothesis that the conversion of PME to PC is catalyzed by a single enzyme in calf brain. In these studies, PME- and PDE-N-methyltransferase activities have been found to be similar, if not identical, with respect to: (1) extractability with Triton X-100; (2) pH optimum; (3) response to divalent cations; (4) apparent Km, for S-adenosyl-l -methionine and KI for S-adenosyl-l -homocysteine, (5) sensitivity to N-ethylmaleimide; and (6) thermal inactivation at 55°. Overall, these results are consistent with the conclusion that in calf brain, PME and PDE are methylated by the same enzyme or by two phospholipid N-methyltransferases having very similar properties. 相似文献
2.
Purification and Characterization of Myosin from Calf Brain 总被引:4,自引:1,他引:3
Mazhar N. Malik Michael D. Fenko Lois Scotto Pat Merz Jeffrey Rothman Helen Tuzio Henryk M. Wisniewski 《Journal of neurochemistry》1983,40(6):1620-1629
Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C. 相似文献
3.
A. Alcázar M. E. Martín E. Soria S. Rodríguez J. L. Fando M. Salinas 《Journal of neurochemistry》1995,65(2):754-761
Abstract: Eukaryotic initiation factor 2B, or guanine nucleotide-exchange factor, has been purified for the first time from the brain by a novel procedure that allows the purification of initiation factor 2 as well and uses a salt wash postmicrosomal supernatant as starting material. The procedure includes a three-part chromatographic step in heparin-Sepharose and in SP-5PW and diethylaminoethyl-5PW ion-exchange high-performance chromatographies. The purification of the factors was followed by measuring activity in the guanine nucleotide-exchange assay and the capacity of initiation factor 2 to form a ternary complex with the initiation form of methionyl-tRNA and GTP. The method yields guanine nucleotide-exchange factor (75%) and highly purified initiation factor 2 (>95%), which are separated in the last step. The exchange factor from the brain is a multimeric protein with five subunits of molecular masses of 82, 65, 52, 42, and 30 kDa; it stimulates ternary complex formation in the presence of GDP, and this activity is inhibited by N -ethylmaleimide. A 37-kDa protein that copurifies with initiation factors is characterized in this study as a new calmodulin-binding protein (p37); it is highly phosphorylated by casein kinase activities and can comigrate with the α subunit of initiation factor 2 under standard sodium dodecyl sulfate electrophoresis conditions. 相似文献
4.
Paraquat-Induced Free Radical Reaction in Mouse Brain Microsomes 总被引:5,自引:0,他引:5
Paraquat has been implicated as an environmental toxin which may induce the syndrome of Parkinson's disease after exposure to this agent. However, the biochemical mechanism by which paraquat causes cell death and neurodegeneration has not been extensively studied. Paraquat was rapidly taken up by nerve terminals isolated from mouse cerebral cortices. It induced lipid peroxidation in a concentration dependent manner in the presence of NADPH and ferrous ion. The maximal stimulation effect was obtained at a paraquat concentration around 100 M and the Kmvalue for paraquat was 46.7 M. The lipid peroxidation required microsomal enzymes. Antioxidants, such as superoxide dismutase, catalase and promethazine significantly inhibited paraquat-induced lipid peroxidation. Due to its structural similarity to the pyridinium compound MPP+(N-methyl-4-phenyl pyridium ion), it may be taken up by dopamine neurons and cause lipid peroxidation and cell death resulting in the manifestation of Parkinsonian syndrome. 相似文献
5.
Ayako Ojima Yasuhito Nakagawa Takayuki Sugiura Yasuo Masuzawa Keizo Waku 《Journal of neurochemistry》1987,48(5):1403-1410
The mechanism involved in the enzymic acylation of 1-[3H]alkylglycero-3-phosphoethanolamine (1-[3H]alkyl-GPE) in brain microsomes was investigated in comparison with the acylation of 1-[3H]alkylglycero-3-phosphocholine (1-[3H]alkyl-GPC). Both the alkyllsophospholipids were acylated without exogenously added cofactors to similar extents. The [14C]arachidonoyl moiety of exogenously added 1-stearoyl-2-[14C]arachidonoyl-GPC was transferred to the alkyllysophospholipids and the transfer was not inhibited by exogenously added free arachidonate. These results indicated that the transferase activity was due to a transacylase that catalyzes the transfer of fatty acids between intact phospholipids. The addition of CoA increased the acylation of 1-[3H]alkyl-GPC two or three times with a high acceptor concentration, and the highest rate of acylation of 1-[3H]alkyl-GPC was observed in the presence of CoA, ATP, and Mg2+. On the other hand, the addition of such cofactors only slightly increased the acylation of 1-[3H]alkyl-GPE. HPLC analysis revealed that docosahexaenoate and arachidonate were transferred to the second position of both [3H]alkyllysophospholipids without cofactors and that other fatty acids were transferred to much lower extents. With the addition of cofactors, the acylation of 1-[3H]alkyl-GPC by both docosahexaenoate and arachidonate increased 1.5-2 times, and high amounts of palmitate, oleate, and linoleate were newly transferred. High amounts of oleate were also transferred to 1-[3H]alkyl-GPE in the presence of cofactors but the acylation by both docosahexaenoate and arachidonate scarcely increased on the addition of these cofactors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Jacques-Andre Maring Richard A. Deitrich Roger Little 《Journal of neurochemistry》1985,45(6):1903-1910
Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes. 相似文献
7.
Kinetic studies showed that under appropriate conditions, [3H]clonidine binds to two distinct receptor sites in calf cortex membranes. At 23 degrees C, binding was obtained at a low-affinity site (dissociation constant, KD = 5.4 nM) and a high-affinity site (KD = 1.1 nM). In contrast, at 0 degree C, selective binding occurred to the low-affinity site only. Consequently, at 0 degree C, it was possible to evaluate the interaction of drugs with the low-affinity receptor directly. On the other hand, competition with the high-affinity receptor could be ascertained by generating displacement curves representing the differential between specific binding values obtained at 23 and 0 degree C. Guanine nucleotides selectively decreased binding to the high-affinity site without apparent influence on the low-affinity [3H]clonidine binding. The activities of various pharmacological agents at the low- and high-affinity clonidine receptors are discussed and compared with WB-4101 binding data. 相似文献
8.
Occurrence of Lipid Peroxidation in Brain Microsomes in the Presence of NADH and Vanadate 总被引:2,自引:0,他引:2
Oxidation of NADH by rat brain microsomes was stimulated severalfold on addition of vanadate. During the reaction, vanadate was reduced, oxygen was consumed, and H2O2 was generated with a stoichiometry of 1:1 for NADH/O2, as in the case of other membranes. Extra oxygen was found to be consumed over that needed for H2O2 generation specifically when brain microsomes were used. This appears to be due to the peroxidation of lipids known to be accompanied by a large consumption of oxygen. Occurrence of lipid peroxidation in brain microsomes in the presence of NADH and vanadate has been demonstrated. This activity was obtained specifically with the polymeric form of vanadate and with NADH, and was inhibited by the divalent cations Cu2+, Mn2+, and Ca2+, by dihydroxyphenolic compounds, and by hemin in a concentration-dependent fashion. In the presence of a small concentration of vanadate, addition of an increasing concentration of Fe2+ gave increasing lipid peroxidation. After undergoing lipid peroxidation in the presence of NADH and vanadate, the binding of quinuclidinyl benzylate, a muscarinic antagonist, to brain membranes was decreased. 相似文献
9.
Purification and Characterization of a Neuropeptide-Degrading Aminopeptidase from Human Brain 总被引:3,自引:2,他引:1
The major aminopeptidase from human post-mortem brain (occipital cortex) was purified to homogeneity (as judged by polyacrylamide gel electrophoresis) by anion-exchange chromatography (two steps) and gel filtration (two steps). The molecular weight of the enzyme was estimated as 105,000 from gel filtration. Maximum activity was obtained in the presence of 0.5 mM Ca2+ and 1 mM 2-mercaptoethanol at pH 7.3. Enzyme activity was lost on freezing and thawing or on lyophilization. The enzyme was inhibited by metal-ion chelating agents, sulphydryl blocking agents, bestatin, and puromycin. A series of amino acyl-7-amido-4-methylcoumarins was hydrolysed by the enzyme, with the alanyl derivative being hydrolysed most rapidly (Km 170 microM). Specificity studies with a series of alanine dipeptides suggested that a hydrophobic second residue favoured hydrolysis. Several naturally occurring neuropeptides, including Leu5-enkephalin (Km 180 microM), cholecystokinin octapeptide, and Arg8-vasopressin, were hydrolysed by the aminopeptidase. In a series of opioid peptides, increasing chain length led to decreased susceptibility to hydrolysis. Sulphation of the Tyr1 residue of Leu5-enkephalin and the Tyr2 residue of cholecystokinin octapeptide made the peptides more resistant to hydrolysis. 相似文献
10.
Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the Kms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the Ki for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM. 相似文献
11.
Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora 总被引:1,自引:1,他引:0
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Erwinia amylovora infected with bacteriophage ERA103 produced an enzyme which degraded the extracellular polysaccharide of noninfected cells. The depolymerase enzyme was purified 15-fold by a procedure which included ammonium sulfate precipitation, ultracentrifugation, CM-Sephadex batchwise separation, Sephadex G-50 column chromatography, and Sephacryl S-200 column chromatography. The enzyme had a molecular weight of approximately 21,000 and a pH optimum of 6.0. Activity was enhanced by supplements of 2-mercaptoethanol or dithiothreitol. 相似文献
12.
Purification, Biochemical Characterization, Binding Activity, and Selectivity of a Glutamate Binding Protein from Bovine Brain 总被引:3,自引:2,他引:1
E. K. Michaelis W. L. Chittenden B. E. Johnson N. Galton C. Decedue 《Journal of neurochemistry》1984,42(2):397-406
A glutamate binding protein was purified from bovine brain to apparent homogeneity. The procedure used for the purification of this protein involved extraction of a crude synaptic membrane fraction with Na-cholate, followed by solubilization of the binding protein from the membranes by Triton X-100, and, finally, affinity batch separation of the protein on L-glutamate-loaded glass fiber. The molecular characteristics of the purified protein were similar to those previously described for the glutamate binding protein from rat brain synaptic membranes and included the following: small Mr (14,000), acidic (pI = 4.7) protein with a single NH2-terminal amino acid (tyrosine), and significant absorption at wave-lengths greater than 300 nm. Complete amino acid analysis of the protein was not achieved, either because of destruction of some amino acids or of incomplete hydrolysis of the protein. The protein bound L-glutamate with high affinity (KD = 0.87 microM), exhibited one class of L-glutamate binding sites, and bound glutamate with a stoichiometry of 0.7 mol ligand/mol protein. The displacement of protein-bound L-glutamic acid by other neuroactive amino acids had characteristics similar to those observed for the displacement of L-glutamate from rat brain synaptic membrane or purified protein binding sites. Finally, the metal ligand formers KCN and NaN3 inhibited the activity of this protein just as they have been shown to do in rat brain synaptic membranes or the purified protein. 相似文献
13.
Neutral and acid sphingomyelinases were copurified from a rat brain P2 fraction by extraction with 1% Triton X-100, followed by (NH4)2SO4 fractionation, acetone powdering, extraction with 1% Triton X-100, (NH4)2SO4 fractionation, Sepharose CL-6B chromatography, and chromatofocusing. The neutral sphingomyelinase was eluted with buffer containing 0.4 M NaCl after the acid sphingomyelinase had been eluted with Polybuffer at pH 5.3. The neutral sphingomyelinase exhibited specific activity of 48,300 nmol/h/mg of protein, with 254-fold purification; the corresponding value for acid sphingomyelinase was 25,300 nmol/h/mg protein, with 668-fold purification from the P2 fraction. The purified neutral sphingomyelinase had no acid sphingomyelinase activity, and vice versa. The properties of the two enzymes were examined. A single band corresponding to a molecular weight of 67,000 was obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for both enzymes. The pI was estimated to be 5.5 for both on isoelectric focusing. The native molecular weights of the neutral and acid sphingomyelinases were found to be 434,000 and 284,000, respectively, on gel filtration with Sepharose CL-6B. The single band obtained for each enzyme on SDS-PAGE was identified as an antigen with antibody raised against the purified neutral sphingomyelinase. Their amino acid compositions were very similar. The neutral and acid sphingomyelinases probably consist of common polypeptides and are immunologically cross-reactive. 相似文献
14.
一种用聚乙二醇制备微粒体的方法 总被引:8,自引:0,他引:8
介绍一种用聚乙二醇(PEG)制备微粒体的方法.大鼠肝匀浆经聚乙二醇-6000凝聚,及两次高速离心即可得到微粒体组分,与超速离心方法比较,可省去超速离心步骤,又缩短了分离制备的时间,是一种比较简单易行的方法. 相似文献
15.
Purification and Characterization of Four NADPH-Dependent Aldehyde Reductases from Pig Brain 总被引:1,自引:1,他引:0
James A. Cromlish Carol K. Yoshimoto T. Geoffrey Flynn 《Journal of neurochemistry》1985,44(5):1477-1484
By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high-Km aldehyde reductase, the low-Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues. 相似文献
16.
Katarzyna Stepniak Agnieszka Girstun Beata Wielgus-Kutrowska Krzysztof Staroń Agnieszka Bzowska 《Nucleosides, nucleotides & nucleic acids》2013,32(6-7):855-859
Calf spleen purine nucleoside phosphorylase (PNP) is considered a model enzyme for the trimeric PNPs subfamily. PCR amplification of the calf phosphorylase from the calf spleen library, cloning, overexpression of the recombinant PNP, its enzymatic activity and interactions with typical ligands of mammalian wild type PNP are described. Relative activity of the recombinant phosphorylase versus several substrates is similar to the respective values obtained for the enzyme isolated from calf spleen. As for the nonrecombinant calf PNP, the unusual fluorescence properties of the PNP/guanine complex were observed and characterized. 相似文献
17.
Hirotaka Yamamoto Hidehiko Konno Teiji Yamamoto Kitae Ito Michinao Mizugaki Yuzo Iwasaki 《Journal of neurochemistry》1987,49(2):603-609
Glutamine synthetase (GS) isolated from human brain formed a single band on sodium dodecyl sulfate-polyacrylamide gel with a molecular weight of 44,000. The enzyme had a specific activity of 179.2 U/mg protein when assayed by measuring the rate of the formation of gamma-glutamylhydroxamate using hydroxylamine as a substrate. In the presence of manganese ions, the relative activity of human brain GS was much lower than that of the sheep brain enzyme. The suppression of activity by increasing the ADP concentration, however, was less marked in the human enzyme than that in the sheep enzyme. Antibodies were raised in rabbits against the purified enzyme. The double-immunodiffusion technique disclosed cross-reactivities among GSs isolated from human, sheep, and rat brains, but the enzymes were not immunologically identical. Immunohistochemically, GS was localized in the cytoplasm of astrocytes in the human and rat brains and in pericentral hepatocytes of the liver. 相似文献
18.
通过离子交换和凝胶过滤层析从湖南产尖吻蝮蛇毒中分离纯化出1个抗补体活性蛋白AACI-I.还原与非还原SDS-PAGE测得其表观分子质量分别为26 kD和22 kD,等电聚焦电泳显示其等电点为pH 8.9.它能抑制补体经典途径和替代途径的溶血,且该作用具有量效和时效性.AACI-I可依次水解纤维蛋白原的Aα、Bβ、γ链,此活性能被EDTA、EGTA和1,10-phenanthroline完全抑制,不受PMSF、SBTI作用影响.同时,该蛋白无精氨酸酯酶活性,提示AACI-I是金属蛋白酶.另外,AACI-I具有azocasein水解活性和水肿活性,当0.01μg/g AACI-I注射小鼠足趾,其诱导肿胀率约为(14.06±6.78)%,而小鼠皮下注射2.6μg/g AACI-Ⅰ后没有发现其有出血毒活性. 相似文献
19.
Ahmed Chadli Jean-Pierre LeCaer Dominique Bladier Raymonde Joubert-Caron Michel Caron 《Journal of neurochemistry》1997,68(4):1640-1647
Abstract: Our previous studies have characterized an endogenous lectin from human brain identified as galectin-1. A soluble ligand of galectin-1 was purified from human brain by affinity chromatography and preparative electrophoresis. The purified ligand (termed HBGp82, for human brain galectin-1-binding polypeptide of 82,000 daltons) has an apparent molecular mass of 82 kDa and is glycosylated by N -linked biantennary complex structures. HBGp82 was partially characterized by microsequencing of peptide fragments. Similar peptides were found in a heat shock of protein of 90,000 daltons, hsp90. However, comparison of apparent molecular weights and matrix-assisted laser desorption mass spectrometry clearly showed that HBGp82 differs to some degree from hsp90. 相似文献
20.
Gening LV Kamensky AA Levitskaya NG Petrochenkov AN Poletaeva II Tarantul VZ 《Biochemistry. Biokhimii?a》2005,70(10):1157-1162
Recent studies performed with crude extracts of mouse tissues showed that the activity of DNA-polymerase iota (Pol iota) can be detected only in brain and testis extracts. To assess whether the activity of Pol iota is associated with animal behavior, we determined Pol iota activity in brain extracts of mice of two lines sharply differing in aggressiveness (RSB and RLB). We found that Pol iota activity in the mice with aggressive behavior was three times higher than in the less aggressive mice. The possible relationship between the activity of Pol iota and animal behavior is discussed. 相似文献