共查询到20条相似文献,搜索用时 125 毫秒
1.
Larry J. Takemoto Jeff S. Hansen Bruce J. Nicholson Michael Hunkapiller Jean-Paul Revel Joseph Horwitz 《生物化学与生物物理学报:生物膜》1983,731(2):267-274
A protein of 26 000 has been shown to be the major component of eye-lens junctions, which are similar but not identical to the gap junctions of liver and other tissues. Cyanogen bromide cleavage of the 26 000 polypeptide from bovine lenses yields a major fragment of 15 000 (fragment 1). However, if the junctions are first treated with trypsin or carboxypeptidase Y, cyanogen bromide treatment yields a fragment of reduced molecular weight. Since protease treatment has been shown to cleave residues almost exclusively from the carboxy-terminal end of the 26 000 polypeptide, it follows that fragment 1 represents the carboxy-terminal half of this molecule, part of which is exposed to proteolytic attack outside the membrane. This latter result is corroborated by the fact that antisera which recognize both the 26 000 polypeptide and fragment 1 fail to do so after preadsorption with intact membranes. In addition, comparative amino acid and partial sequence analyses of the 26 000 polypeptide and fragment 1 indicate that fragment 1 is more hydrophilic in character, suggesting that much of the amino-terminal half of the 26 000 polypeptide is buried within the lipid bilayer. 相似文献
2.
According to previous authors, cytochrome , when extracted from bovine liver by a detergent method, is called cytochrome . On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome .Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, , decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome , whereas does not change for cytochrome and cytochrome . This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome , because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature. 相似文献
3.
The N-terminal cyanogen bromide fragment from the Bβ chain of bovine fibrinogen was isolated, and its molecular weight was estimated to be approximately 14,000–15,500. The ratio of the Michaelis-Menten constants, , for its hydrolysis by bovine thrombin was found to be 3 × 10?7 [(NIH unit/liter)s]?1, indicating that the Bβ fragment is a poor substrate for thrombin compared to the corresponding Aα chain fragment. This value of is too small to account for the rate of release of fibrinopeptide B from fibrinogen by thrombin. It is suggested that, while the Aα chain contains all of the amino acid residues necessary to interact with thrombin, the Bβ chain does not; i.e., some of the binding sites that are used in the hydrolysis of the Bβ chain are assumed to be located on either the α or γ chains of fibrinogen. An alternative hypothesis is that, after the Bβ chain fragment is removed from the fibrinogen molecule, it does not have the proper conformation to be hydrolyzed by thrombin. 相似文献
4.
Charge-pulse current-relaxation studies have been performed with lipid bilayer membranes in the presence of the hydrophobic ion dipicrylamine. From the analysis of the relaxation times and amplitudes the translocation rate constant of dipicrylamine as well as the partition coefficient β between membrane surface and water could be evaluated. In a first series of experiments membranes made from monoolein or dioleoylphosphatidylcholine in a number of different solvents were studied, as well as virtually solvent-free bilayer membranes made from monolayers. The thickness of the hydrocarbon layer of these membranes varied between 5.0 and 2.5 nm. While β was almost insensitive to variations in , a strong decrease of with increasing membrane thickness was found; the observed dependence of on approximately agreed with the theoretically expected influence of membrane thickness on the height of the dielectric barrier. No specific differences between Mueller-Rudin films and solvent-free (Montal-Mueller) membranes other than differences in thickness were found. In a further series of experiments the chemical structure of the lipid was systematically varied (number and position of double bonds in the hydrocarbon chain, nature of the polar head group). The translocation rate constant was much larger in phosphatidylethanolamine membranes than in phosphatidylcholine membranes. A strong increase of was found when the number of double bonds in the hydrocarbon chain was increased from one to three. These changes were discussed in terms of membrane fluidity and dielectric barrier height. Much higher values of were observed in lipids with ester linkage between hydrocarbon chain and glycerol backbone, as compared with the corresponding ether analogs. This finding is qualitatively consistent with determinations of dipolar potentials in monolayers of ester and ether lipids. When cholesterol is added to phosphatidylcholine membranes, the translocation rate constant increases up to five-fold, while the partition coefficient β remains virtually constant. The variation of in this case can be largely accounted for by a decrease in membrane thickness and a concomitant reduction in dielectric barrier height. In membranes made from the negatively charged lipid phosphatidylserine the partition coefficient of dipicrylamine strongly increased with ionic strength, as expected from the Gouy-Chapman theory of the surface potential. 相似文献
5.
Dorothy A. Schafer Donald E. Hultquist 《Biochemical and biophysical research communications》1981,100(4):1555-1561
A protease which generates a soluble hemepeptide from bovine liver microsomal cytochrome has been isolated from the membrane fraction of rabbit reticulocytes. Inhibition by pepstatin and an acidic pH optimum indicate that the protease belongs to the acid protease class. Little cytochrome -processing activity is observed in rabbit erythrocytes. We suggest that the protease may be involved in the processing which generates the proteins of the methemoglobin reduction system from their membrane-bound precursors during the maturation of the erythroid cell. 相似文献
6.
Michel Hughes Daniéle Duval Heidy Schmid Patrick Kitabgi Michel Lazdunski Jean-Pierre Vincent 《Life sciences》1982,31(5):437-443
This paper describes the interaction of apamin, the bee venom neurotoxin, with its receptor in the guinea pig colon. The pharmacological activity of the toxin was assayed by measuring its contracting effect on guinea pig colon preparations that had been previously relaxed by neurotensin. The IC50 value of apamin in this bioassay is 7 nM. These pharmacological data are compared to the binding properties of apamin to smooth muscle membranes prepared from guinea pig colon. The highly radiolabeled monoiododerivative of apamin binds to its colon receptor with a dissociation constant . The maximal binding capacity of colonic membranes is 30dfmol/mg of protein. The dissociation constant of the unmodified toxin is 23 pM. The difference between the toxin concentrations that produce half-maximal effects in the binding and pharmacological studies arises from the different experimental conditions used for the two assays. 相似文献
7.
8.
Phosphate transporter of bovine heart mitochondria was purified by solubilization of submitochondrial particles with octylglucoside and fractionation of the extract with ammonium sulfate. After reconstitution into liposomes the purified protein catalyzed phosphate transport which was sensitive to mersalyl and other SH reagents. Transport measured either as or exchange was proportional to protein concentration and time. The but not the exchange was stimulated several fold by valinomycin plus nigericin in the presence of K+. The reconstituted system provides a suitable assay during purification of the mitochondrial phosphate transporter. 相似文献
9.
10.
Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N4-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C3), -A-U-G-A- (mutant C4) and -A-U-A-A- (mutant ) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C3), 3.2 (C4) and 0.3 (). The finding that mutant C3 no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C4 RNA bound more efficiently than wild type, and that mutant RNA showed substantial ribosome binding capacity whereas the single mutant C3 did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon. 相似文献
11.
A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ()-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [3H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated ), there are several minor surface glycopeptides () which are apparent extrinsic membrane components, and two surface glycopeptides () which are intrinsic membrane components. 相似文献
12.
The presence of cytoplasmic poly(A) polymerase has been established in sea urchin eggs and four-cell embryos by subcellular fractionation and use of enucleate egg halves. ATP is the only ribonucleoside triphosphate incorporated. This incorporation is time dependent, contingent on input protein concentration, and immune to a variety of antimetabolites known to inhibit DNA-directed RNA synthesis. Both the unfertilized egg and the four-cell embryo cytoplasmic poly(A) polymerase activities display a preference for Mn2+. While oligo(A)4 is inactive as a primer, addition of and stimulates ATP incorporation. On a unit per milligram protein basis, the endogenous activity associated with cytoplasmic fractions obtained from nucleate and enucleate egg halves is 36 and 83% that obtained with the cytoplasmic fraction prepared from the unfertilized egg. In the presence of , both the nucleate and enucleate egg halves exhibit 81% of the activity associated with the unfertilized egg cytoplasmic fraction. The level of Mn2+ cytoplasmic poly(A) polymerase activity from the four-cell embryo is approximately 50% that of the unfertilized egg. This decrease does not appear to be due to either a postfertilization alteration in the subcellular localization of poly(A) polymerase or an increase in RNase activity. Supplementation with failed to restore the four-cell embryo cytoplasmic poly(A) polymerase potential to a level comparable to that of the unfertilized egg. Suppression of postfertilization protein synthesis by emetine, however, prevents this developmental decline in ATP incorporation thereby suggesting that postfertilization cytoplasmic poly(A) polymerase activity is subject to negative translational control. 相似文献
13.
Bernard Lacour Tilman Drüeke Evelyne Pierandreï Bernadette Nabarra Jean-Louis Funck-Brentano 《生物化学与生物物理学报:生物膜》1981,648(2):151-161
Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na+ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na+ efflux rate constant () of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na+ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca2+-free medium, bovine PTH lost its capacity to inhibit (). Thus, the presence of extracellular Ca2+ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on , bovine PTH induced no change in net Na+ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na+ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca2+ in the incubation medium to be operative. 相似文献
14.
Walter C. Schneider 《Biochemical and biophysical research communications》1977,74(4):1607-1612
Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a while 65% of the particulate (microsomal) DNA reassociated with a Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences. 相似文献
15.
José Remacle 《生物化学与生物物理学报:生物膜》1980,597(3):564-576
The in vitro incorporation of cytochrome into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome than did microsomal preparations; 60% of this cytochrome could not be reduced by the NADH-cytochrome reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome . These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell. 相似文献
16.
Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte (). The concentration dependence of the binding to ghosts reveals two saturable components. [14C]Flufenamate binds with high affinity () to 8.5·105 sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity () to a second set of sites (4.6·107 per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [14C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [14C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [14C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [14C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein. 相似文献
17.
If the bicyclic peptide ring proposed by Gross . (1,2) does in fact exist in nisin and related antibiotics, then the unusual β-methyllanthionine component must be significantly distorted from its conformation in the free state, as determined by x-ray structure analysis. The torsion angles about the SCβ bonds are 50–100° from the torsion angles in models of the sulfur-bridged peptide ring proposed for nisin. The chirality of the methylated β-carbon atom is (S). The conformation of the amino acid differs from that of -lanthionine only by a 180° rotation of a carboxyl group about the bond. 相似文献
18.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
19.
John L. McGregor Kenneth J. Clemetson Elizabeth James Phillippe Clezardin Marc Dechavanne Ernst F. Lüscher 《生物化学与生物物理学报:生物膜》1982,689(3):513-522
Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (), apparent molecular weight (), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP132–1354–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same but different were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins. 相似文献
20.
W.McD. Armstrong W.R. Bixenman K.F. Frey J.F. Garcia-Diaz M.G. ORegan Jeanie L. Owens 《生物化学与生物物理学报:生物膜》1979,551(1):207-219
Na+, K+ and Cl? concentrations () and activities (), and mucosal membrane potentials () were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na+ and Cl? and 5.4 mM K+. Na+ and K+ concentrations were determined by atomic absorption spectrometry and Cl? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO3. 14C-labelled inulin was used to determine extracellular volume. was measured with conventional open tip microelectrodes, with solid-state Cl?-selective silver microelectrodes and and with Na+- and K+-selective liquid ion-exchanger microelectrodes. The average recorded was ?34 mV. , and were 51, 105 and 52 mM. The corresponding values for , and were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na+ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K+ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl?. significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na+ and Cl? is implicated in intracellular Cl? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na+ and Cl? electrochemical potential differences (Δμ̄Na and Δμ̄Cl). Δμ̄Na (?7000 J · mol?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ̄Cl (1000–2000 J · mol?1). 相似文献