共查询到20条相似文献,搜索用时 15 毫秒
1.
Previous reports from this laboratory and others have established that both the rabbit and human erythrocyte membranes contain multiple protein kinase and phosphate acceptor activities. We now report that these membranes also contain phosphoryl acceptor sites for the soluble cyclic AMP-dependent and -independent protein kinases from rabbit erythrocytes. The rabbit erythrocyte membrane, which does not contain a cyclic AMP-dependent protein kinase, has at least four polypeptides (Bands 2.1, 2.3, 4.5, and 4.8) which are phosphorylated in the presence of the soluble cyclic AMP-dependent protein kinases I, IIa, and IIb isolated from rabbit erythrocyte lysates. The resulting phosphoprotein profile is very similar to that obtained for the cyclic AMP-mediated autophosphorylation of human erythrocyte membranes. The activities of the soluble cyclic AMP-dependent protein kinases toward the membranes have been studied at several pH values. Although the substrate specificity of the three kinases is similar, polypeptide 2.3 appears to be phosphorylated to a greater extent by kinase IIa than by I or IIb. This occurs at all pH values studied. Also apparent is that the pH profile for membrane phosphorylation is different from that of histone phosphorylation. The phosphorylation of membrane proteins can also be catalyzed by the soluble erythrocyte casein kinases. These enzymes are not regulated by cyclic nucleotides and can use either ATP or GTP as their phosphoryl donor. Polypeptides 2.1, 2.9, 4.1, 4.5, 4.8, and 5 of both human and rabbit erythrocyte membranes are phosphorylated in the presence of GTP and the casein kinases. This reaction is optimal at pH 7.5. Experiments were performed to determine whether the phosphorylation of the membranes by the soluble and membrane-bound kinases is additive or exclusive. Our results indicate that after maximal autophosphorylation of the erythrocyte membranes, phosphoryl acceptor sites are available to the soluble cyclic AMP-dependent and -independent protein kinases. Furthermore, after maximal phosphorylation of the membranes with one type of soluble kinase, further 32P incorporation can occur as a result of exposure to the other type of soluble kinase. 相似文献
2.
Edon Melloni Franca Salamino Bianca Sparatore Mauro Michetti Sandro Pontremoli 《Archives of biochemistry and biophysics》1982,216(2):495-502
Human erythrocytes are shown to contain soluble proteinase(s) that convert excess β-hemoglobin introduced by in vitro entrapment to modified forms that are bound to the erythrocyte membrane. The bound modified hemoglobin chains are degraded in the membrane to yield acid soluble products. Native hemoglobin chains are not bound to the membrane and are not degraded. The cooperative degradation of excess β-hemoglobin chains by soluble and membrane-bound enzymes occurs at neutral pH and does not require energy. The results provide a role for the membrane-bound acid proteinases. 相似文献
3.
Effect of phospholipase C, trypsin and neuraminidase on binding of bilirubin to mammalian erythrocyte membranes. 总被引:1,自引:0,他引:1
M K Ali S Tayyab 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2001,129(2-3):355-362
Binding of bilirubin to erythrocyte membranes of human, buffalo, sheep and goat was studied after phospholipase C, trypsin and neuraminidase treatment. Phospholipase C and trypsin treatment of membranes greatly enhanced the bilirubin binding in all mammalian species, whereas, neuraminidase treatment resulted into a small increase in the membrane-bound bilirubin. Human erythrocyte membranes bound the highest amount of bilirubin, whereas buffalo, sheep and goat erythrocyte membranes showed different mode of bilirubin binding. The order of bilirubin binding to unmodified as well as neuraminidase-treated erythrocyte membranes was: human>sheep>buffalo>goat; the order was: human>buffalo>sheep>goat; in phospholipase C- and trypsin-treated erythrocyte membranes. These binding results indicate that membrane phospholipids are directly involved in the interaction of bilirubin with the membranes as the differences observed in the membrane-bound bilirubin among mammalian species were directly correlated with the sum of choline phospholipids, especially phosphatidylcholine and sphingomyelin content of the erythrocyte membranes. The negatively charged phosphate moiety of phospholipids of the membranes appears to inhibit a large amount of bilirubin binding to the membrane as its removal by phospholipase C greatly enhanced the binding. Furthermore, membrane proteins and carbohydrate also seem to play a significant regulatory function on the binding as their degradation and/or removal in the form of glycopeptides by trypsin expose a large number of bilirubin binding sites. 相似文献
4.
Carl G. Gahmberg Georg Taurn Ismo Virtanen Jorma Wartiovaara 《Journal of cellular biochemistry》1978,8(3):337-347
Human erythrocyte membranes of the En(a–) blood group lack the major sialoglycoprotein (glycophorin). By absorption of a crude antiglycophorin antiserum with En(a–) membranes a specific antiglycophorin antiserum was obtained. By immune electron microscopy we showed that glycophorin is randomly distributed on the surface of normal erythrocytes. When polycationized ferritin, which mainly binds to glycophorin, was used as a marker a similar even labeling of normal erythrocyte membranes was seen. En(a–) membranes bound much less of this marker. In freeze-fracturing the intramembrane particles of both membrane types had a similar distribution and appeared in equal amounts. However, partial removal of spectrin from these membranes, followed by incubation at pH 6 resulted in more extensive aggregation of the particles in En(a–) membranes than in normal membranes. The results may be interpreted as glycophorin contributing by electrostatic repulsion to the random distribution of the intramembrane particles in normal cells. This repulsion is weakened in En(a–) cells by the lack of glycophorin. 相似文献
5.
Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes 总被引:2,自引:0,他引:2
Effect of pH and temperature on the binding of bilirubin to human erythrocyte membranes was studied by incubating the membranes at different pH and temperatures and determining the bound bilirubin. At all pH values, the amount of membrane-bound bilirubin increased with the increase in bilirubin-to-albumin molar ratios (B/As), being highest at lower pH values in all cases. Further, linear increase in bound bilirubin with the increase in bilirubin concentration in the incubate was observed at a constant B/A and at all pH values. However, the slope value increased with the decrease in pH suggesting more bilirubin binding to membranes at lower pH values. Increase in bilirubin binding at lower pH can be explained on the basis of increased free bilirubin concentration as well as more conversion of bilirubin dianion to monoanion. Temperature dependence of bilirubin binding to membranes was observed within the temperature range of 7 degrees -60 degrees C, showing minimum binding at 27 degrees C and 37 degrees C which increased on either side. Increase in bilirubin binding at temperatures lower than 20 degrees C and higher than 40 degrees C can be ascribed to the change in membrane topography as well as bilirubin-albumin interaction. 相似文献
6.
Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg2+.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group. 相似文献
7.
H J Lustig V Nussenzweig R S Nussenzweig 《Journal of immunology (Baltimore, Md. : 1950)》1977,119(1):210-216
Immunoglobulin (Ig) is associated with erythrocyte membranes during infection of A/J mice with Plasmodium berghei. It was detected by agglutination of the cells with rabbit antibodies to mouse IgG and by a radioimmunoassay with 125I-labelled rabbit antibodies to mouse IgG. As shown by the degree of agglutination and of binding of radiolabeled antibodies to the erythrocyte membranes, the amount of Ig increased with time after infection and paralleled parasitemia and reticulocytosis. The erythrocyte-associated immunoglobulins are mainly IgG but IgM was also present on the cells of some mice. A large proportion of the Ig could be eluted at 37 degrees C and was analyzed by immunoprecipitation and acrylamide gel electrophoresis. A radioautographic study with 125I-labeled anti-mouse IgG revealed that both parasitized and nonparasitized reticulocytes of infected mice had much larger amounts of membrane-bound immunoglobulin than did mature, nonparasitized erythrocytes. The nature of the bonds between the Ig and the surface membrane of the reticulocytes is not known. The Ig could be part of immune complexes nonspecifically bound to the cell surface. However, since we have not detected Fc or C3d receptors on reticulocytes, it is possible that the Ig constitutes autoantibodies against reticulocytes or antibodies against parasitic antigens present on the cell membrane. 相似文献
8.
Most of the chicken erythrocyte's sphingomyelin is hydrolyzed when the chicken red blood cells are incubated in hypotonie solution at 37 °C.Addition of detergents, such as Triton X-100 or Na-cholate, is essential for hydrolysis of external [3H ]sphingomyelin by the erythrocyte membranes.Pure plasma membranes show relatively high sphingomyelinase activity while no activity could be detected in the soluble fraction of the cells. Mg2+ and Mn2+ activate the enzyme while Ca2+ and EDTA strongly inhibit its activity. The optimal pH of the membrane-bound sphingomyelinase lies between pH 7.0–9.0. The detergents Triton X-100 and Na-cholate, at concentrations of 0.5% () solubilize the membrane-bound enzyme. Human erythrocytes fail to exhibit sphingomyelinase activity.The correlation between the sphingomyelinase activity and its localization is discussed. 相似文献
9.
The autophosphorylation of rabbit and human erythrocyte membranes has been studied under various experimental conditions. The phosphopeptides of the erythocyte membranes were identified using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by ratioautography. The pattern of phosphorylatiion of membrane components differs with respect to the phosphoryl donor used (ATP or GTP) and to the pH at which the reaction is carried out. Both species appear to contain at least two distinct membrane-bound protein kinases. The human erythrocyte membrane contains a cyclic adenosine 3'5'-monophosphate (cyclic AMP)-dependent protein kinase and several substrates for this kinase. Only ATP can be used as a phosphoryl donor for this kinase. In contrast, the rabbit erythrocyte membrane does not contain a cyclic AMP dependent protein kinase but does contain a kinase which utilizes only ATP as the phosphoryl donor and is specific for certain endogenous substrates at low pH. Both the human and rabbit erythrocyte membranes contain a kinase which utilizes GTP, perhaps also ATP, as the phosphoryl donor. The substrates of these kinases are similar in both species. 相似文献
10.
6-Phosphofructo-1-kinase (phosphofructokinase) (ATP:D-fructose-6-P 1-phosphotransferase, EC 2.7.1.11) can be identified in sheep heart homogenates in two forms, a soluble form and a form bound to the particulate fraction. Homogenates from immediately-dissected hearts have the enzyme in the soluble form, while those collected after a delay have the enzyme bound to the particulate fraction. Aldolase appears to show the same change in its location. Homogenization in a solution with concentrated macromolecular species (20% albumin) results in a greater association of phosphofructokinase and of aldolase to the particulate fraction in homogenates from immediately dissected hearts. Phosphofructokinase activity can be solubilized by two specific means: by high ionic strength, which is dependent upon specific salts; or by low ionic strength, which is dependent upon the presence of phosphofructokinase substrates or modifier ligands. These two means of solubilization are affected differently upon decreasing the pH below 6.9: the solubilization at low ionic strength is prevented, whereas phosphofructokinase is still solubilized by high ionic strength. Under the latter condition, the enzyme is in the inactive dimeric state, which can be activated at an alkaline pH. Myofibrils present in the particulate fraction can account for the binding of phosphofructokinase in heart homogenates. Purified myofibrils, when added to heart supernatant fluids, can bind phosphofructokinase at a slightly acidic pH. Conditions for phosphofructokinase binding to myofibrils, as well as its dissociation, follow what was observed with the binding of phosphofructokinase to the particulate fraction. At an acidic pH, and in the presence of a high concentration of ATP, phosphofructokinase exhibits low activity. However, if phosphofructokinase is assayed under these conditions while bound to myofibrils, the enzyme is activated. 相似文献
11.
The role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase from rat liver Golgi membranes during the acute-phase response. 总被引:5,自引:1,他引:4
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Golgi-membrane-bound Gal beta 1-4GlcNAc alpha 2-6-sialyltransferase (CMP-N-acetylneuraminate:beta-galactoside alpha 2-6-sialyltransferase, EC 2.4.99.1) behaves as an acute-phase reactant increasing about 5-fold in serum in rats suffering from inflammation. The mechanism of release from the Golgi membrane is not understood. In the present study it was found that sialyltransferase could be released from the membrane by treatment with ultrasonic vibration (sonication) followed by incubation at reduced pH. Maximum release occurred at pH 5.6, and membranes from inflamed rats released more enzyme than did membranes from controls. Galactosyltransferase (UDP-galactose:N-acetylglucosamine galactosyltransferase; EC 2.4.1.38), another Golgi-located enzyme, which does not behave as an acute-phase reactant, remained bound to the membranes under the same conditions. Release of the alpha 2-6-sialyltransferase from Golgi membranes was substantially inhibited by pepstatin A, a potent inhibitor of cathepsin D-like proteinases. Inhibition of release of the sialyltransferase also occurred after preincubation of sonicated Golgi membranes with antiserum raised against rat liver lysosomal cathepsin D. Addition of bovine spleen cathepsin D to incubation mixtures of sonicated Golgi membranes caused enhanced release of the sialyltransferase. Intact Golgi membranes were incubated at lowered pH in presence of pepstatin A to inhibit any proteinase activity at the cytosolic face; subsequent sonication showed that the sialyltransferase had been released, suggesting that the proteinase was active at the luminal face of the Golgi. Golgi membranes contained a low level of cathepsin D activity (EC 3.4.23.5); the enzyme was mainly membrane-bound, since it could only be released by extraction with Triton X-100 or incubation of sonicated Golgi membranes with 5 mM-mannose 6-phosphate. Immunoblot analysis showed that the transferase released from sonicated Golgi membranes at lowered pH had an apparent Mr of about 42,000 compared with one of about 49,000 for the membrane-bound enzyme. Values of Km for the bound and released enzyme activities were comparable and were similar to values reported previously for liver and serum enzymes. The work suggests that a major portion of sialyltransferase containing the catalytic site is released from a membrane anchor by a cathepsin D-like proteinase located at the luminal face of the Golgi and that this explains the acute-phase behaviour of this enzyme. 相似文献
12.
Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis. 相似文献
13.
Sztiller M Puchala M Kowalczyk A Bartosz G 《Redox report : communications in free radical research》2006,11(6):263-271
The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation. 相似文献
14.
Human erythrocyte membranes which had been thoroughly extracted with organic solvents contained 20 nmol of fatty acids/mg dry wt. The major fatty acids were palmitic and stearic with their monoethenoic derivatives as minor constituents. No other fatty acids were detected. When solvent-extracted membranes were digested with Pronase about 90% of the original content of fatty acids was retained in the insoluble residue. Fatty acids were linked to membrane proteins through alkali-labile bonds of which 30% were of a thiol ester and the remainder of an O-ester type. This conclusion is based on differential liberation of fatty acids by hydroxylamine at pH 7.0 and pH 11.0. Two extracts of membranes enriched in peripheral proteins (bands 1, 2, 5 and 2.1, 4.1, 4.2, 6) were prepared and extracted with organic solvents but each contained about six times less fatty acids than the parent solvent-extracted membranes. Glycophorin A contains little if any covalently bound fatty acids. Anion transporter (band 3) contains about 1 mol of thiol ester of fatty acid. This accounts for about half of the thiol ester-linked fatty acids in the parent solvent-extracted membranes. Most of the O-ester-linked fatty acids are linked to an undisclosed membrane protein. 相似文献
15.
M D Kurski? T P Kondratiuk A A Osipenko A N Fedorov V A Grigor'eva 《Biokhimii?a (Moscow, Russia)》1982,47(1):34-42
The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000. 相似文献
16.
Summary X- and gamma-irradiation of human erythrocyte membranes (250–1000 Gy) was found to decrease the ratio of weakly to strongly immobilized signal height of membrane-bound maleimide spin label (Mal-6). Subsequent incubation of spin-labeled membranes at ambient temperature (21 °C) induced a progressive increase in this ratio, faster for membranes irradiated with low doses which was hampered by protease inhibitors. These results demonstrate that ionizing radiation stimulates proteolysis of erythrocyte membrane proteins by membrane-associated proteases. 相似文献
17.
Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte 总被引:3,自引:0,他引:3
The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion. 相似文献
18.
《Redox report : communications in free radical research》2013,18(6):263-271
AbstractThe aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H2O2; ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation. 相似文献
19.
Identification of the pH-dependent membrane anchor of carboxypeptidase E (EC 3.4.17.10) 总被引:9,自引:0,他引:9
Carboxypeptidase E (CPE), a peptide hormone-processing enzyme, is present within secretory granules in both a soluble form and a form which is membrane-bound at pH 5.5 but soluble at neutral pH. Antisera raised against a peptide corresponding to the predicted COOH-terminus of CPE bind to the membrane-associated form of CPE but not to the soluble form. This COOH-terminal region is predicted to form an amphiphilic alpha-helix, containing several pairs of hydrophobic residues separated by hydrophilic residues. Synthetic COOH-terminal peptides 11-24 residues in length are able to bind to bovine pituitary membranes and can be extracted by conditions that extract the membrane-bound form of CPE. The influence of pH on the membrane binding of a 21-residue COOH-terminal peptide is similar to the membrane binding of CPE: at pH values less than 6 the majority of the peptide is membrane-bound, while at pH values above 8 less than 20% is membrane-bound. Both the 21-residue COOH-terminal peptide and the purified membrane form of CPE, but not the soluble form, partition into Triton X-114 only at low pH (pH less than 6). Combined polar and hydrophobic interactions of the COOH-terminal peptide appear to be responsible for the reversible, pH-dependent association of CPE with membranes. 相似文献
20.
The quaternary structure of an active form of rabbit muscle phosphofructokinase was studied by sedimentation and electron microscopy. Active enzyme centrifugation studies at pH 7.0 and 23 +/- 1 degrees C showed that phosphofructokinase sediments as a single component with a sedimentation coefficient of 12.2 +/- 0.5 S. Identical results were obtained in two assay and three solvent systems. Boundary sedimentation studies of phosphofructokinase in the presence of 1.0 mM fructose 6-phosphate, 0.1 mM adenylyl imidodiphosphate at pH 7.0 and 23 +/- 1 degrees C were performed. The results showed that the sedimentation coefficient of phosphofructokinase remains constant within the range of protein concentration studied and assumes a value of 12.4 S. The molecular weights of the subunit and the 12.4 S component were measured by sedimentation equilibrium yielding values of 83,000 and 330,000 for the monomeric and polymeric species, respectively. It is, therefore, concluded that the active form of phosphofructokinase is indeed the tetrameric species. The structure of the phosphofructokinase tetramer was also studied by electron microscopy of negatively stained specimens. Particles identified as tetramers measured approximately 9 nm in diameter by 14 nm in length. The observed size and shape are consistent with the hydrodynamic measurements. Structural features within the tetramer were interpreted as due to the four individual subunits, each one approximately 4 X 6 X 6 nm in size, arranged with D2 symmetry. 相似文献