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J W Gurd

Publications and source records attributed to J W Gurd.

80 records · Page 5Linked to original sources
PubMed1973

Purification and properties of a mouse liver plasma-membrane glycoprotein hydrolysing nucleotide pyrophosphate and phosphodiester bonds.

1. A mouse liver plasma-membrane preparation was solubilized in an N-dodecylsarcosinate-Tris buffer, pH7.8, and the proteins and glycoproteins were separated by a rate-zonal centrifugation in sucrose-detergent gradients. 2. A peak of alkaline phosphodiesterase activity which sedimented ahead of the 5'-nucleotidase peak was associated with a major glycoprotein component of the plasma membrane. 3. The phosphodiesterase activity was then purified further by gel filtration and gave a single glycoprotein band after electrophoresis on polyacrylamide gels. The apparent molecular weight of the polypeptide at pH7.4 and 8.9 was 128000-130000 and was independent of the polyacrylamide concentration. Electrophoresis in gels containing deoxycholate showed that the protein band was coincident with phosphodiesterase activity. 4. After two-dimensional immunoelectrophoresis, with agarose containing rabbit anti-(mouse plasma-membrane) antiserum as second dimension, the enzyme showed one component which was also coincident with the phosphodiesterase activity. 5. An amino acid composition of the glycoprotein is presented. Carbohydrate analysis indicated the presence of glucosamine, neutral sugars and sialic acid. 6. The enzyme was also a nucleotide pyrophosphatase, as shown by a similar enrichment during purification of activity towards ATP, NAD(+), UDP-galactose and UDP-N-acetylglucosamine. The phosphodiesterase activity, measured by using dTMP p-nitrophenyl ester as substrate, was competitively inhibited by nucleotide pyrophosphate substrates. The enzyme showed little or no activity towards RNA, cyclic AMP, AMP, ADP and glycerylphosphorylcholine. 7. The significance of this enzyme activity in the plasma membrane is discussed.

Amino Acids↗
PubMed1973

Immunochemical characterization of proteins from mouse liver plasma membranes.

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an (125)I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate-1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5'-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate-Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.

Animals↗
PubMed1972

Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents.

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.

Acrylates↗
PubMed1972

Preparation and properties of nexuses and lipid-enriched vesicles from mouse liver plasma membranes.

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.

Amino Acids↗
PubMed1972

The distribution of surface antigens during fractionation of mouse liver plasma membranes.

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.

Animals↗
PubMed1971

Biosynthesis of liver membranes. Incorporation of ( 3 H)leucine into proteins and of ( 14 C)glucosamine into proteins and lipids of liver microsomal and plasma-membrane fractions.

1. The smooth-and rough-microsomal and the light and heavy plasma-membrane fractions of mouse liver homogenates were prepared and characterized by using biochemical markers. 2. The hexosamine/protein ratio was threefold higher in the plasma membranes than in the smooth-microsomal fraction. Glucosamine was bound only to protein, and galactosamine was attached mainly to lipids. 3. [(3)H]-Leucine and [(14)C]glucosamine were injected into animals and the rates of incorporation of radioactivity into the fractions were determined. Both precursors were rapidly incorporated into the microsomal fractions, but plasma membranes showed a slower rate of synthesis which reached a maximum at 2-4h after intravenous administration. 4. The light- and heavy-plasma-membrane fractions showed similar patterns of incorporation, and therefore a precursor-product relationship appears unlikely. 5. Plasma membranes, especially the light subfraction, showed appreciable incorporation of hexosamine into chloroform-methanol-soluble components which were shown to be mainly glycolipids. 6. The results indicate that liver plasma-membrane proteins and glycoproteins are synthesized at similar rates. However, glycolipid synthesis in plasma membranes occurred more rapidly.

Animals↗