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1.
The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.  相似文献   

2.
Calmodulin-binding proteins in chromaffin granule membrane and chromaffin cell plasma membranes have been investigated and compared. Chromaffin granules were purified by centrifugation over a 1.7 M sucrose layer. Plasma membranes were obtained in a highly purified form by differential and isopycnic centrifugation. Enzymatic determinations of 5'-nucleotidase, a generally accepted plasma membrane marker, showed a 40-50-fold enrichment as compared to the cell homogenate. Marker enzyme studies demonstrated only minimal contamination by other subcellular organelles. After solubilization with Triton X-100, calmodulin-binding proteins were isolated from chromaffin granule membranes and plasma membranes by affinity chromatography on a calmodulin/Sepharose 4B column. On two-dimensional polyacrylamide gelelectrophoresis a prominent protein (Mr = 65,000, pI ranging from 5.1 to 6) consisting of multiple spots, was present in the calmodulin-binding fraction from chromaffin granule membranes as well as from plasma membranes. Besides this 65 kDa protein both fractions had at least four groups of proteins in common. Also, proteins typical for either preparation were observed. In the calmodulin-binding protein preparations from chromaffin granule membranes a prominent spot with Mr = 80,000 and a pH ranging from 5.0 to 5.7 was present. This protein was enzymatically and immunologically identified as dopamine-beta-monooxygenase.  相似文献   

3.
The properties of particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from purified rabbit skeletal muscle membrane fragments were studied. Four membrane fractions were prepared by sucrose gradient centrifugation and the fractions characterized by analysis of marker enzymes. Guanylate cyclase activity was highest in the fraction possessing enzymatic properties typical of sarcolemma, while fractions enriched with sarcoplasmic reticulum had lower activities. In the presence of suboptimal Mn2+ concentrations, Mg2+ stimulated particulate guanylate cyclase activity both before and after solubilization in 1% Triton X-100. Guanylate cyclase activity was biphasic in the presence of Ca2+. Increasing the Ca2+ concentration from 10(-8) to 10(-5) M decreased the specific activity. As the Ca2+ concentration was further increased to 5 . 10(-4) M enzyme activity again increased. After solubilization of the membranes in 1% Triton X-100, Ca2+ suppressed enzyme activity. Studies utilizing ionophore X537A indicated that the altered effect of Ca2+ upon the solubilized membranes was independent of asymmetric distribution of Ca2+ and Mg2+.  相似文献   

4.
Nitrobenzylthioinosine, a potent nucleoside-transport inhibitor, binds to high-affinity sites on the human erythrocyte membrane. This binding is a specific interaction with functional nucleoside-transport sites. The protein(s) responsible for high-affinity nitrobenzylthioinosine binding was purified 13-fold by treatment of haemoglobin-free 'ghosts' with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with Triton X-100 and passage of the soluble extract through a DEAE-cellulose column equilibrated with Triton X-100. Void-volume fractions were collected and treated with Bio-Beads SM-2 to remove detergent. These fractions contained 31% of the starting nitrobenzylthioinosine-binding activity. They also contained D-glucose-sensitive cytochalasin B-binding activity. Nitrobenzylthioinosine binding to the partially purified preparation was saturable (apparent Kd 1.6 nM) and inhibited by nitrobenzylthioguanosine, dipyridamole and uridine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pooled void-volume fractions revealed the presence of only two detectable protein bands, the broad zone 4.5 (containing glucose-transport protein) and a small amount of band 7.  相似文献   

5.
Plasma membranes were isolated from the cultured Sertoli cells of 20-day-old rat testes by differential centrifugation and sucrose density fractionation. The distribution and purity of subcellular components was determined by marker enzyme analysis of gradient fractions. The plasma membrane fraction showed an enrichment in two plasma membrane marker enzymes, 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase-specific activities, of 9- and 23-fold, respectively. Forty-two percent and 52% of the total cellular 5'-nucleotidase and ouabain-sensitive Na+/K+-ATPase activities, respectively, were found in the membrane fraction. The protein yield of plasma membrane was approximately 6% of the total cellular protein. Two-dimensional polyacrylamide gel electrophoresis was used to compare [35S] methionine- and [3H] glucosamine-labeled membrane proteins. The incorporation of [35S] methionine and [3H] glucosamine was increased in several proteins when the cultured Sertoli cells were treated with follicle-stimulating hormone, insulin, retinol, and testosterone. Isolated Sertoli cell membranes contained a membrane-associated form of plasminogen activator. Analysis of this plasminogen activator demonstrated that the membrane-associated enzyme existed primarily as a single 38,000-40,000-Mr form.  相似文献   

6.
Two highly purified plasma membrane fractions have been obtained from mouse parotid glands by a combination of differential centrifugation and isopycnic centrifugation in discontinuous sucrose gradients. The membranes were characterized by enzymic, chemical and morphological criteria. The effect of isoproterenol, which induces parotid acinar cells to proliferate, upon sialic acid and five different enzyme activities located in the plasma membrane phosphodiesterase (EC 3.1.4.1), Mg2+-ATPase (EC 3.6.1.4), leucine aminopeptidase (EC 3.4.1.1), protein kinase (EC 2.7.1.37) and sialyltransferase (EC 2.4.99.1), were quantified along the cell cycle. Plasma membrane sialic acid content falls 30% within 30 min and remains depressed for at least 6 h with the major restoration towards normal levels occurring between 12 and 16 h later. In contrast multiple daily isoproterenol injections lead to a more than 2-fold elevation of sialic acid content. Sialyltransferase activity rises 2-fold by 12 h after isoproterenol treatment and then rapidly falls. This enzyme has a pH optimum of 6.5, requires a divalent cation for activity and is inhibited by Triton X-100. Other enzyme activities showed markedly different changes after isoproterenol stimulation, either increasing, decreasing or remaining unaltered. These continuous functional modifications suggest an active role of the plasma membrane in the control of the proliferative cycle.  相似文献   

7.
Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.  相似文献   

8.
Large amounts of injected radiolabeled low density lipoproteins have been found by others to accumulate primarily in the liver and studies in various types of isolated cells, including hepatocytes, have indicated the presence of specific cell membrane recognition sites for lipoproteins. In the present studies, the high affinity binding of radiolabeled low density lipoproteins ([125I]LDL, d 1.020--1.063 g/mL) was measured in the major subcellular fractions of porcine liver homogenates. The nuclear and mitochondrial fractions were 1.9- and 1.4-fold enriched in binding activity with respect to unfractionated homogenates and contained 15% and 12% of the total binding activity, respectively. The microsomes, which contained most of the plasma membranes and endoplasmic reticulum, were approximately 4-fold enriched in binding and contained 73% of the binding activity. Microsomal subfractions obtained by differential homogenization and centrifugation procedures were 5.6--7.0-fold enriched in LDL binding and contained 54--58% of the homogenate binding activity. They were separated by discontinuous sucrose density gradient centrifugation into fractions which contained "light" and "heavy" plasma membranes and endoplasmic reticulum. The heavy membrane fraction was 2--4 fold in binding with respect to the parent microsomes (16--22 fold with respect to the homogenate). There was no enrichment of binding activity in the other two fractions. Two plasma membrane "marker" enzymes, nucleotide pyrophosphatase and 5'-nucleotidase, were also followed. Of the two, binding in the sucrose density gradient subfractions most closely followed nucleotide pyrophosphatase, which was also most highly enriched (3.2--3.3-fold) in the heavy membrane fraction, but did not follow it exactly. The enzyme was 2-fold richer in the light membranes than in the parent microsomes, though the light membrane binding activity was only 0.4--1.4 times that of the parent microsomes. High affinity binding was time and temperature dependent, saturable, and inhibited by unlabeled low density lipoproteins but not by unrelated proteins. Binding was stimulated 2--3 fold Ca2+, was not affected by treatment with Pronase or trypsin and was inhibited by low concentrations of phospholipids and high density lipoproteins (HDL). Heparin-Mn2+ treatment of HDL did not affect its ability to inhibit [125I] LDL binding. The LDL recognition site was distinct from the liver membrane asialoglycoprotein receptor; LDL binding was not inhibited by desialidated fetuin. We conclude that porcine liver contains a high affinity binding site that recognizes features common to both pig low density and high density lipoproteins. Further studies may elucidate the significance of this binding site in lipoprotein metabolism.  相似文献   

9.
The effects of phospholipids on the properties of hepatic 5'-nucleotidase   总被引:1,自引:0,他引:1  
Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.  相似文献   

10.
Plasma membranes from hamster liver were prepared by differential and continuous sucrose gradient centrifugation. The membranes contained a low Km cyclic AMP phosphodiesterase (EC 3.5. lc) and calmodulin. The activity of the membrane phospho-diesterase was reduced with EGTA and LaCl3. The membrane low Km cyclic AMP phosphodiesterase was solubilized with Triton X-100 and then chromatographed on DEAE-cellulose to remove calmodulin. After elution, phosphodiesterase was stimulated with exogenous calmodulin; this activation was blocked with EGTA. Thus a low Km cyclic AMP phosphodiesterase has been shown to be dependent on calmodulin for “maximal” activity.  相似文献   

11.
The outer membrane of Campylobacter coli, C. jejuni and C. fetus cell envelopes appeared as three fractions after sucrose gradient centrifugation. Each outer membrane fraction was contaminated with succinate dehydrogenase activity from the cytoplasmic membrane fraction. Similarly the inner membrane fraction was contaminated with 2-ketodeoxyoctonate and outer membrane proteins including the porin(s). The separation of these two membranes was not facilitated by variations in lysozyme treatment, cell age, presence or absence of flagella, or longer lipopolysaccharide chain length. Sodium lauroyl sarcosinate extraction resulted in an outer membrane fraction which contained some inner membrane contamination and produced multiple bands upon sucrose gradient centrifugation. Triton X-100 extraction removed the inner membrane from the outer membrane and Triton X-100/EDTA treatment extracted lipopolysaccharide-rich regions of the outer membrane which contained almost exclusively the Campylobacter porin(s). These data indicated that the inner and outer membranes of the Campylobacter cell envelope were very difficult to separate, possibly because of extensive fusions between these two membranes.  相似文献   

12.
The activity of UDPgalactose-asialo-mucin galactosyltransferase (EC 2.4.1.74) in microsomal and Golig subfractions was stimulated 2.4-fold after disruption of the membrane permeability barrier by hypotonic incubation. In the presence of Triton X-100, galactose transfer to asialo-mucin was increased 12-fold in rough microsomes and 5-fold in smooth microsomes both with and without hypotonic incubation; while in the Golgi subfractions no stimulation by detergent was observed. These experiments indicate differences in enzyme-lipid or enzyme-protein interactions in microsomes and Golgi membranes. Furthermore, these results strongly support the conclusion that the UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal fractions is not due to contamination by Golgi vesicles but represents an enzyme activity endogenous to the endoplasmic reticulum.  相似文献   

13.
We developed a method for extracting raft-like, liquid-ordered membranes from the particulate fraction prepared from porcine trachealis smooth muscle. This fraction, which contains most of the plasma membrane in this tissue, was homogenized in the presence of cold 0.5% Triton X-100. After centrifugation, membranes containing high contents of sphingomyelin (SM) and cholesterol and low phosphatidylcholine (PC) contents remained in the pellet. Thirty-five millimolar octyl glucoside (OG) extracted 75% of these membranes from the Triton X-100-resistant pellet. These membranes had low buoyant densities and accounted for 28% of the particulate fraction lipid. Their lipid composition, 22% SM, 60% cholesterol, 11% phosphatidylethanolamine, 8% PC, <1% phosphatidylinositol, and coisolation with 5'-nucleotidase and caveolin-1 suggest that they are liquid-ordered membranes. We compared characteristics of OG and Triton X-100 extractions of the particulate fraction. In contrast to Triton X-100 extractions, membranes released from the particulate fraction by OG were mainly collected in low buoyant fractions at densities ranging from 1.05 to 1.11 g/ml and had phospholipid and cholesterol contents consistent with a mixture of liquid-ordered and liquid-disordered membranes. Thus, OG extraction of apparent liquid-ordered membranes from Triton X-100-resistant pellets was not due to selective extraction of these membranes. Low buoyant density appears not to be unique for liquid-ordered membranes.  相似文献   

14.
Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.  相似文献   

15.
Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg2+-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.  相似文献   

16.
Lipoxygenase activity was measured in human platelet subcellular fractions. From a sonicated platelet preparation, a granule fraction, mixed membranes (surface and intracellular) and cytosol fractions were separated by differential centrifugation. With respect to activities in the sonicated preparation, the lipoxygenase was slightly enriched in both the cytosol and mixed-membrane fractions and consistently de-enriched in the granule fractions. Approx. 65% and 20% of the total cell enzyme activity were found in the cytosol and mixed membranes respectively, with only 8% present in the granule fraction. Additionally we measured the lipoxygenase activity in purified surface- and intracellular-membrane subfractions prepared from the mixed membranes by free-flow electrophoresis. There was a slight enrichment in activity in the intracellular membrane fraction compared with that in the mixed membranes, and a depletion of activity in the surface membranes. Characterization of the enzyme activity, i.e. time course, pH-dependence, Ca2+-dependence, Vmax. and Km for arachidonic acid, and the carbon-position specificity for this acid, failed to reveal any significant differences between the membrane-bound and soluble forms of the lipoxygenase. These findings suggest that in human platelets the same lipoxygenase is associated with the membranes as in the cytosol and that the membrane-bound activity predominates in intracellular membrane elements.  相似文献   

17.
Although protein kinase C, an enzyme dependent on calcium, phospholipid and diacylglycerol, has been found in high levels in ovarian tissues, its biologic function is yet unknown. In initial studies on the role of this enzyme in regulating ovarian functions, we compared protein kinase C activity in subcellular fractions of porcine corpora lutea and medium follicles. Highest protein kinase C-specific activities were found in the cytosol, followed by microsomes and mitochondria for both follicles and luteal tissues. Solubilization of all membrane-containing fractions by 0.2% Triton X-100 was required for full expression (a 4-fold average increase) of protein kinase activity. Extraction of membrane fractions with 0.5 M NaCl or sonication in a hypotonic medium revealed that 90% of the total mitochondrial protein kinase C activity and 50% of the microsomal activity was tightly membrane-bound. Characterization of both cytosolic and Triton X-100 extracted membrane preparations of luteal tissue by diethylaminoethyl (DEAE)-cellulose chromatography revealed a single peak of protein kinase C activity eluting at 80 mM NaCl. Cytosolic fractions of corpora lutea contained 3 times more protein kinase C-specific activity than did cytosolic fractions of follicles. In contrast, mitochondria from medium follicles contained 30% more specific protein kinase C activity than did luteal mitochondria. These higher cytosolic levels of protein kinase C-specific activity in corpora lutea suggest that the enzyme may play an important role in the process of luteinization or in the regulation of luteal function.  相似文献   

18.
The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.  相似文献   

19.
A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.  相似文献   

20.
The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.  相似文献   

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