Immunochemical characterization of proteins from mouse liver plasma membranes.
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ABSTRACT:
SUBMITTER: Gurd JW
PROVIDER: S-EPMC1165901 | biostudies-other | 1973 Dec
REPOSITORIES: biostudies-other
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Project description:1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20-25% was soluble in 50mm-sodium carbonate-bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30-35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.
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Project description:Human plasma fibronectin has been purified by a non-denaturing affinity chromatography procedure [Vuento & Vaheri, (1979) Biochem.J. 183, 331--337], and antisera have been raised by immunizing rabbits with the native protein. The antisera reacted strongly with native fibronectin, but only weakly with reduced and alkylated fibronectin or with heat-denaturated fibronectin. Denaturation also affected the haemagglutinating and gelatin-binding activities of fibronectin and increased its susceptibility to proteolytic degradation. The antisera reacted with fragments of fibronectin obtained by proteolysis with plasmin. Large fragments (mol.wt. 180000--200000), lacking the region harbouring the interchain disulphide bridges but containing the sites responsible for gelatin-binding and haemagglutinating activity, showed as intense a reaction with the antisera as intact fibronectin. Smaller peptides showed a weaker reaction. All fragments tested showed sensitivity to denaturation in their reaction with the antisera. The results were interpreted as showing that: (1) native fibronectin has an ordered conformation that is easily perturbed by denaturation; (2) most of the antigenic determinants of the protein are dependent on conformation; (3) the region of the fibronectin molecule containing the interchain disulphide bridges has only few antigenic determinants; and (4) covalent interaction of the two subunits does not contribute to the antigenic structure recognized by rabbit antisera. The observed correlation between the antigenic activity and a structural and functional intactness of fibronectin suggests that the antibodies to native fibronectin could be used as a conformational probe in studies on this protein.
| S-EPMC1162272 | biostudies-other
Project description:1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.
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Project description:1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified gamma-globulin antibody fraction was iodinated with (125)I. The reaction of the (125)I-labelled gamma-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The gammaglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ;Light' and ;heavy' mouse liver plasma-membrane subfractions bound similar amounts of gamma-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO(3)-Na(2)CO(3) buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.
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Project description:Extraction of mouse liver plasma membranes with 4% (w/v) N-laurylsarcosinate-tris buffer, pH7.8, solubilized 80-90% of the protein and 60% of the 5'-nucleotidase activity. The membrane residue remaining after extraction was resolved on sucrose gradients into two fractions: a vesicular membrane fraction and a fraction characterized by the presence of large numbers of nexuses in an amorphous background. The vesicular fraction had a phospholipid/protein weight ratio of 7:1, it contained most of the plasma-membrane glycolipids, and polyacrylamide-gel electrophoresis indicated the presence of only five to eight proteins, including two or three glycoproteins. The 5'-nucleotidase and leucine naphthylamidase specific activities were 23- and 6-fold higher respectively than in the plasma membranes. Electron microscopy of thin sections and negatively stained preparations indicated that the nexuses present in the second fraction closely resembled gap junctions present in tissue sections and isolated plasma membranes. The nexus fraction contained a distinctive protein pattern, and of the 20 proteins present about four were identified as glycoproteins by Schiff-periodate staining. Examination of the lipid composition of the fractions by t.l.c. showed that in the nexus fraction, phospholipids and glycolipids were present in small amounts compared with triglycerides and cholesterol. Amino sugar analyses confirmed the t.l.c. results and amino acid analysis showed the fractions to have characteristic protein compositions. A ;reconstituted' membranous fraction prepared by dialysis against MgCl(2) of membrane components soluble in N-laurylsarcosinate-tris buffers, pH7.8, lacked the trilaminar image characteristic of the two other membrane fractions isolated and was devoid of enzyme activities. The results indicate that proteins and glycoproteins play an important role in the structural maintenance of the nexuses isolated from the liver by the present procedure.
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Project description:The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.
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Project description:Filaggrin (FLG) and corneodesmosin (CDSN) are two key proteins of the human epidermis. FLG loss-of-function mutations are the strongest genetic risk factors for human atopic dermatitis. Studies of the epidermal distribution of canine FLG and CDSN are limited. Our aim was to better characterize the distribution of FLG and CDSN in canine skin. Using immunohistochemistry on beagle skin, we screened a series of monoclonal antibodies (mAbs) specific for human FLG and CDSN. The cross-reactive mAbs were further used using immunoelectron microscopy and Western blotting. The structure of canine CDSN and FLG was determined using publicly available databases. In the epidermis, four anti-FLG mAbs stained keratohyalin granules in the granular keratinocytes and corneocyte matrix of the lower cornified layer. In urea-extracts of dog epidermis, several bands corresponding to proFLG and FLG monomers were detected. One anti-CDSN mAb stained the cytoplasm of granular keratinocytes and cells of both the inner root sheath and medulla of hair follicles. Dog CDSN was located in lamellar bodies, in the extracellular parts of desmosomes and in corneodesmosomes. A protein of 52 kDa was immunodetected. Genomic DNA analysis revealed that the amino acid sequence and structure of canine and human CDSN were highly similar.
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Project description:Nitrobenzylthioinosine (NBMPR) was employed as a probe of the nucleoside transporters from rat and guinea-pig liver. Purified liver plasma membranes prepared on self-generating Percoll density gradients exhibited 16-fold (rat) and 10-fold (guinea pig) higher [3H]NBMPR-binding activities than in crude liver homogenates (3.69 and 14.7 pmol/mg of protein for rat and guinea-pig liver membranes respectively, and 0.23 and 1.47 pmol/mg of protein for crude liver homogenates respectively). Binding to membranes from both species was saturable (apparent Kd 0.14 and 0.63 nM for rat and guinea-pig membranes respectively) and inhibited by uridine, adenosine, nitrobenzylthioguanosine (NBTGR) and dilazep. Uridine was an apparent competitive inhibitor of high-affinity NBMPR binding to rat membranes (apparent Ki 1.5 mM). There was a marked species difference with respect to dipyridamole inhibition of NBMPR binding (50% inhibition at 0.2 and greater than 100 microM for guinea-pig and rat respectively). These results are consistent with a role of NBMPR-binding proteins in liver nucleoside transport. Exposure of rat and guinea pig membranes to high-intensity u.v. light in the presence of [3H]NBMPR resulted in the selective radio-labelling of membrane proteins which migrated on sodium dodecyl sulphate/polyacrylamide gels with apparent Mr values in the same range as that of the human erythrocyte nucleoside transporter (45 000-66 000). Covalent labelling of these proteins was abolished when photolysis was performed in the presence of non-radio-active NBTGR as competing ligand.
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