共查询到20条相似文献,搜索用时 31 毫秒
1.
Shigenobu Nakamura 《生物化学与生物物理学报:生物膜》1976,426(2):339-347
The 5′-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5′-nucleotidase exhibited the same properties as the 5′-nuelcotidase in plasma membranes. The 5′-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and . The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate. 相似文献
2.
A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5′-nucleotidase inhibitor α,β-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5′-nucleotidase. An antibody against mouse liver 5′-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5′-nucleotidase and the necessity for distinguishing between true 5′-nucleotidase and non-specific phosphatase activity is discussed. 相似文献
3.
The properties of the methylphosphonyl group as a substrate analog for the phosphoryl moiety of various biological phosphoryl donors have been investigated in several enzymic phosphoryl transfer reactions. The synthesis and characterization of adenosine 5′-[β-methylphosphonyl]diphosphate, adenosine 5′-methylphosphonate, acetyl methylphosphonate, and methylphosphonoenolpyruvate are fully described. Adenosine 5′-[β-methylphosphonyl]diphosphate is not a substrate for adenylate kinase, hexokinase, 3-phosphoglycerate kinase, glycerol kinase, phosphofructokinase, creatine kinase, alkaline phosphatase, or nucleoside 5′-diphosphate kinase. Competitive inhibition of ATP was observed with hexokinase and 3-phosphoglycerate kinase with . Adenosine 5′-methylphosphonate was a substrate for adenylate deaminase and 5′-nucleotidase, but not for adenylate kinase, acid phosphatase, 5′-phosphodiesterase, or 3′-phosphodiesterase. Acetyl methylphosphonate inhibits the reaction of acetyl phosphate with acetate kinase, but methylphosphonoenolpyruvate has no effect upon the reaction of phosphoenolpyruvate with pyruvate kinase. The results indicate that with the exception of 5′-nucleotidase, the methyphosphonyl group is incapable of undergoing phosphoryl transfer. One interpretation among others is that a metaphosphate-type mechanism is required for these processes. 相似文献
4.
About 15% of the total (3H)leucine incorporated into protein by isolated rat liver mitochondria could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids. 相似文献
5.
Thymidylate synthetase from amethopterin-resistant is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP. 相似文献
6.
Alexander S. Sun James F. Holland Kant Lin Takao Ohnuma 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(4):577-584
5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro. 相似文献
7.
Dimers of escherichia coli F' factors 总被引:1,自引:0,他引:1
Covalently closed circular DNA dimers of several sex factors have been isolated. One of these, F′451, a dimer of F′450, has a molecular weight of 230 × 106 daltons. F′451 (λ) containing a λ prophage has a molecular weight of 260 × 106 and is probably the largest covalent closed circle of DNA yet reported. These dimers arise spontaneously and are of unknown origin and significance. 相似文献
8.
Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [3H] tRNA extracted from B grown in the presence of [5,6-3H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [3H] tRNA. Neither [5-3H]-uridine, [5,6-3H]-UTP, nor [5,6-3H]-poly U released tritium in the present assay conditions. 相似文献
9.
A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104–125 (1972)) is characterized by high levels of glucose-6-phosphatase and 5′-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes and as well as NADPH- and NADH-cytochrome reductase activities are present but at lower levels than found in microsomes. Cytochrome oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5′-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside trisphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus. 相似文献
10.
The non-histone chromatin proteins of growing and non-growing tissues were compared by two-dimensional polyacrylamide gel electrophoresis. The tissues studied were normal rat liver, regenerating rat liver, thioacetamide-treated rat liver, normal rat kidney, Novikoff hepatoma and Walker 256 carcinosarcoma. Although most of the protein components were common to all of the tissues studied, the densities and sizes of spots C18, CP, C21, C25, CQ, CR, CS and CT were greater in the growing tissues than in the non-growing tissues, including the thioacetamide-treated liver. In the latter, the increased densities and sizes of spots C18, C21 and CQ are presumably related to the markedly increased nucleolar size rather than to cell division. Accordingly the increases in sizes and densities of spots C25, CP, CR, CS and CT are apparently of importance to the growth processes of normal and tumor tissues. The number of tissue specific proteins was small compared with the number of proteins in this fraction and includes BP and CBL for normal liver, BJ′ for kidney and CG′, CH′ and CPfor the tumors. 相似文献
11.
Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, /, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver is presented. 相似文献
12.
In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats 总被引:1,自引:0,他引:1
R J Martin 《Life sciences》1974,14(8):1447-1453
Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. incorporation of acetate-14C into adipose tissue lipid was not significantly different, however, liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat. 相似文献
13.
Alfred H. Merrill Kihachiro Horiike Donald B. McCormick 《Biochemical and biophysical research communications》1978,83(3):984-990
Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The Ki, 3 μM, is considerably lower than the Km for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The Ki determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than Ki, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the rate of pyridoxal 5′-phosphate formation. 相似文献
14.
incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes. 相似文献
15.
T Kanamori T Hayakawa T Nagatsu 《Biochemical and biophysical research communications》1974,57(2):394-398
Tolbutamide at a concentration of 10 mM inhibited cyclic AMP-dependent protein kinase in cell-free preparations of rat parotid glands as reported in rat adipose tissues. Incubation of rat parotid slices with 10 mM tolbutamide markedly interfered with the isoproterenol stimulation of amylase secretion. A carboxy derivative of tolbutamide, 1-butyl-3--carboxyphenylsulfonylurea, had minimal inhibitory effects both on protein kinase activity and on amylase secretion. These evidences strongly suggest the participation of cyclic AMP-dependent protein kinase in amylase secretion. 相似文献
16.
In vivo regulation of hepatic protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation 总被引:2,自引:0,他引:2
O Sudilovsky 《Biochemical and biophysical research communications》1974,58(1):85-91
administration of glucagon caused an increase in the dissociation of protein kinase subunits which was accompanied by elevated adenosine 3′,5′-monophosphate concentrations in the rat liver. Concomitantly, there was a decrease in non saturated adenosine 3′,5′-monophosphate binding sites. A reduction in protein kinase activity measured in the presence of the cyclic nucleotide was apparent at 5 minutes of glucagon administration while enzyme activity assayed in the absence of adenosine 3′,5′-monophosphate was already increased after one minute. Glucose, given through an intragastric tube, caused no changes in the effect of glucagon on hepatic protein kinase. 相似文献
17.
Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ( 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized. 相似文献
18.
19.
Patricia A. Dennis Nathan N. Aronson 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,798(1):14-20
The uptake and degradation of a homologous rat serum asialoglycoprotein, 125I-asialoorosomucoid, and the effects on this metabolism by leupeptin, a proteinase inhibitor, were studied in the perfused rat liver. 125I-Asialoorosomucoid was rapidly taken up by the liver () and acid-soluble degradation products began to appear in the circulating perfusate medium after 20–30 min. These products accounted for 60–65% of the initially added radioactivity after 90 min of perfusion. The early events in the galactose-mediated uptake of 125I-asialoorosomucoid were unchanged by the presence of leupeptin. However, the appearance of acid-soluble degradation products was greatly reduced when livers had been pretreated with the inhibitor (1.0 mg for 60 min). This effect corresponded with an increase in acid-precipitable material being located within the lysosomal-rich fraction from homogenates of leupeptin-treated livers. Leupeptin inhibited degradation of 125I-asialoorosomucoid by approx. 85% relative to control values over 90 min of perfusion. Inhibition of asialoorosomucoid degradation was also demonstrated in vitro. Leupeptin (1.0 mM) reduced hydrolysis of this glycoprotein substrate by greater than 50% during a 24 h incubation with isolated lysosomal enzymes. The thiol proteinases, cathespin B, H and L, which are known to be inhibited by leupeptin, are apparently involved in initiating digestion of rat 125I-asialoorosomucoid within liver lysosomes. As a result of inhibition by leupeptin both in the perfused liver and in vitro very limited changes occured in the native molecular weight of the starting glycoprotein. 相似文献
20.
W T Shier 《Biochemical and biophysical research communications》1977,75(1):186-193
Acyl coenzyme A:lysolecithin acyltransferase plays a major role in regulating the amount of lysolecithin in cell membranes. The acyltransferase activity in microsomal preparations from rat liver, rat heart and rabbit gastric mucosa is inhibited by a series of tertiary amine local anesthetics, detergents, and some inhibitors of cyclic nucleotide phosphodiesterases. Aspirin and indomethacin cause elevated lysolecithin/lecithin ratios in the stomachs of mice after oral administration. Inhibition of acyltransferase activity in microsomal preparations by local anesthetics correlates with reported anesthetic potencies at approximately reported therapeutic dosages. In BHK-13 cells acyltransferase activity is inhibited at to the concentrations that have been reported to cause alterations in the mobility and topography of cell surface receptors. 相似文献