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PubMed1982

Synthesis and assembly of membrane glycoproteins. Membrane anchoring COOH-terminal domain of vesicular stomatitis virus envelope glycoprotein G contains fatty acids.

Two polypeptides associated with the envelope of vesicular stomatitis virus are obtained by exhaustive proteolytic digestion of the virion. Analysis of the tryptic peptides and determination of the partial amino acid sequence show that the larger membrane-anchoring peptide is derived from the hydrophobic COOH terminus of the viral transmembrane glycoprotein G. The smaller peptide is, however, derived from the nonglycosylated matrix protein M. Analysis of the membrane-anchoring peptide fragments obtained from virus labeled with [3H]palmitic acid shows that the larger peptide fragment contained all the fatty acid present in G, suggesting that the fatty acids in conjunction with the hydrophobic domain may be involved in the binding of G protein to the membrane.

Amino Acid Sequence↗

PubMed1982

Comparison of receptor properties of erythrocyte membrane glycoproteins.

Membrane glycoproteins from horse, sheep, goat and bovine erythrocytes were solubilized and purified. These glycoproteins could be placed in three groups based on their degrees of glycosylation: The major bovine erythrocyte glycoprotein (BGII) had 77% sugar, the minor bovine glycoprotein (BGI) had 27% sugar and the others had approximately 50% sugar. Four of the glycoproteins aggregated in a uniform way in aqueous solution--one, BGII, did not. Four had similar subunit sizes of 25-34,000 daltons, but BGII was larger--55,000 daltons. Receptor functions (for plant and invertebrate lectins, antibodies and cell surfaces) of these glycoproteins were primarily those dependent upon the presence of terminal sialic acid residues.

Animals↗

PubMed1982

Estrogen-induced changes in chick oviduct membrane glycoproteins.

Membrane protein and glycoprotein changes during estrogen-induced differentiation of the chick oviduct were studied. Membrane preparations from chicks treated with diethylstilbestrol for 3, 6, 10, and 20 days; from chicks treated with hormone for 10 days and then withdrawn for 2 days; and from mature hens were incubated with either GDP-[14C]Man or GDP-[3H]Man. Double label analysis of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that labeling of proteins with apparent molecular weights of 91,000 and 57,000 is enhanced upon diethylstilbestrol treatment. The effect is reversed upon hormone withdrawal. Analysis of [14C]Man-proteins by SDS-PAGE and fluorography reveals at least 18 labeled proteins, confirms the double label gel results, and shows changes in three additional proteins. Estrogen-induced changes in membrane proteins and glycoproteins were also studied by Coomassie blue staining, periodic acid-Schiff staining, and autoradiography of 125I-concanavalin A binding following SDS-PAGE. Changes are observed in at least 9 Coomassie blue, 7 periodic acid-Schiff and 13 125I-concanavalin A bands. Some proteins are enhanced by estrogen treatment and are diminished upon withdrawal, whereas other proteins are diminished by estrogen treatment and are enhanced by withdrawal. Hen oviduct membrane preparations were labeled with combinations of UDP-GlcNAc, GDP-Man, and UDP-Glc. Tunicamycin inhibits incorporation of [3H]Man and [14C]GlcNAc into protein by 84% and 65%, respectively. SDS-PAGE reveals that transfer of GlcNAc to a 36,000 protein is the least sensitive to tunicamycin. SDS-PAGE of proteins labeled by incubation with GDP-[14C]Man and UDP-[3H]Glc indicate that the presence of Glc does not alter the typical Man-protein-labeling pattern and processing of Glc from individual acceptors uniform.

Acetylglucosamine↗

PubMed1975

The participation of lipid-linked oligosaccharide in synthesis of membrane glycoproteins.

Membrane preparations from hen oviduct catalyze the transfer of mannose from GDP-mannose into three components: mannosyl phosphoryl polyisoprenol, oligosaccharide-lipid, and glycoprotein. Eivence that mannosyl phosphoryl polyisoprenol serves as a mannosyl donor for synthesis of both oligosaccharide-lipid and glycoproteins was previously reported (Waechter, C.J., Lucas, J.J., and Lennarz, W.J. (1973) J. Biol. Chem. 248, 7570-7579). In this study the oligosaccharide-lipid has been isolated, and the oligosaccharide has been partially characterized. Based on paper chromatography the oligosaccharide chain contains 7 to 9 glycose units. The glycose at the reducing terminus is N-acetylglucosamine, whereas mannose is found at the nonreducing end. When UDP-N-acetyl[14C]glucosamine is incubated with oviduct membranes in the absence of GDP-mannose, a 14C-labeled chitobiosyl lipid, but little oligosaccharide-lipid is synthesized. When GDP-mannose is also present in the incubation mixture an oligosaccharide-lipid is formed containing N-acetyl[14C]glucosaminyl residues. This oligosaccharide-lipid is chromatographically identical with the [14C]mannose-containing oligosaccharide-lipid isolated in the earlier study cited above. When the N-acetyl[14C]glucosamine-oligosaccharide released from the oligosaccharide-lipid by mild acid is treated with partially purified alpha-mannosidase the major radioactive product is [14C]chitobiose. Evidence that the [14C]mannose-containing oligosaccharide-lipid serves as an oligosaccharide donor for glycoprotein synthesis was obtained by incubation of partially purified oligosaccharide-lipid with the membranes. The products of this incubation were shown to be glycoproteins on the basis of their sensitivity to pronase, as determined by both gel filtration and paper electrophoresis. Similar experiments, using oligosaccharide-lipid doubly labeled with [14C]mannose and N-acetyl[3H]glucosamine, provided evidence that the oligosaccharide chain of the oligosaccharide-lipid is transferred en bloc to glycoprotein s.

Acetamides↗

PubMed1984

Induction of tolerance by haptenated liposomes carrying mouse erythrocyte membrane glycoprotein.

Membrane sialoglycoprotein (GP) of mouse erythrocytes were incorporated into artificial membranes of liposomes which also contained DNP-aminocaproylphosphatidylethanolamine hapten. The liposomes were tested for their potential to modulate the anti-hapten antibody response in mice as a plausible model of haptenated isologous erythrocytes which is known to be a potent tolerogen. Repeated intravenous inoculation with DNP-liposomes carrying mouse GP resulted in a significant suppression of splenic plaque-forming cells and serum antibody produced in the recipient mice against the subsequent immunization with DNP-KLH in saline or DNP-HGG in Freund's complete adjuvant, in a strictly hapten-specific fashion. The suppression was long-lasting, and the secondary response to DNP-KLH given over a month later was also affected. DNP-liposomes without GP, or those with rabbit GP, exhibited little or no suppressive effect. The observed capacity of mouse GP-bearing DNP-liposomes was reminiscent of that of haptenated mouse erythrocytes in that only the IgG response, but not the IgM response, was suppressed.

Animals↗

PubMed1986

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes.

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane on self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label in secondary lysosomes increased by first order kinetics (k = [56 min]-1) from less than 0.1% (background level) to a steady-state level of approximately 2.5% of the total label. As analyzed by NaDodSO4 PAGE, labeled molecules of Mr 160-190 kD were depleted and of Mr 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all Mr classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constituents to secondary lysosomes is a limited and selective process, and that only approximately 1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

Animals↗

PubMed1993

Correlation between membrane glycoprotein and detergent dissected membrane protein in the assessment of LMIF: a report of 51 ALA cases.

Assessment of 51 amoebic liver abscess cases for leukocyte migration inhibition factor released using membrane glycoprotein and detergent dissected membrane protein (DDMP) of axenic Entamoeba histolytica (NIH:200). Lymphokines release by T lymphocytes in response to purified amoebic membrane glycoprotein (PAMG) against whole amoebic lysate (WAL), dissect out protein against whole amoebic lysate and membrane glycoprotein against dissected protein was tested by leukocyte migration inhibition test on blood samples from proved amoebic liver abscess cases. A significant increase was noted in the release of lymphokines and 100% positivity was observed with both PAMG and DDMP compared to 78% with whole amoebic lysate. The difference between means leukocyte migration indices of the membrane glycoprotein and whole amoebic lysate, detergent dissected protein and whole amoebic lysate with regards to release LMIF were found to be highly significant (P < 0.001), (P < 0.005) respectively. But insignificant difference and very much similarity was noted between the means of membrane glycoprotein and dissect out protein sensitized T lymphocytes with regards to lymphokine release in vitro. This shows the patients had high degree of leukocyte sensitized to pure amoebic membrane glycoprotein and detergent dissected membrane protein compared to whole amoebic lysate. These findings indicate that detergent dissected protein has similar antigenicity with membrane glycoprotein in elicitation cell mediated immune response in amoebic liver abscess cases.

Animals↗

PubMed1989

Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma.

Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L-fucose and the core sugar D-mannose turned over with much shorter half-lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life-span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.

Animals↗

PubMed1984

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection.

In the first paper of this series (Bennett et al., 1984), light-microscope radioautographic studies showed that colchicine or vinblastine inhibited intracellular migration of glycoproteins out of the Golgi region in a variety of cell types. In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered.

Animals↗

PubMed1986

Covalent attachment of sulfhydryl-specific, electron spin resonance spin-labels to Fab' fragments of murine monoclonal antibodies that recognize human platelet membrane glycoproteins. Development of membrane protein specific spin probes.

A general method for the production of high-affinity, nitroxide-labeled, protein-specific spin probes is described in this paper. Fab' fragments are generated from protein-specific, murine monoclonal antibodies by pepsin digestion and mild reduction with cysteine. The free sulfhydryl group located in the carboxy-terminal region of these molecules and produced de novo by this manipulation is then alkylated by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), thereby generating spin-labeled Fab' fragments of these monoclonal antibodies. Two prototypic monoclonal antibodies were tested, each specific for a different integral membrane glycoprotein of human blood platelets. The results indicate that Fab' spin probes generated by this method retain the ability to bind to these glycoproteins within the membrane of intact platelets. These reagents thus represent probes that can be generally used to monitor integral membrane protein mobility on the surface of the intact cell.

Animals↗

PubMed1988

Derived protein sequence, oligosaccharides, and membrane insertion of the 120-kDa lysosomal membrane glycoprotein (lgp120): identification of a highly conserved family of lysosomal membrane glycoproteins.

The 120-kDa lysosomal membrane glycoprotein (lgp120) is an acidic, heavily glycosylated membrane protein enriched in the lysosomal membrane. To determine the basis for its selective transport to and stability in lysosomes, we have investigated the structure of lgp120. By using an oligonucleotide probe corresponding to the amino terminus of rat lgp120, we isolated and characterized cDNA clones containing the entire coding region. The deduced amino acid sequence demonstrates that lgp120 contains a putative signal peptide, 18 sites for N-linked glycosylation, a single membrane-spanning segment, and a short (11 amino acid) cytosolic tail. The sequence suggests a distinct domain organization, with two luminal glycosylated regions separated by a nonglycosylated proline-rich region. Proteolysis in detergent showed that the protein was not intrinsically resistant to exogenous or endogenous proteases. The N-linked oligosaccharides on lgp120, tetraantennary structures with two lactosamine repeats on one of the branches, were not different from those of glycoproteins on the plasma membrane. lgp120 was similar in its domain organization and portions of its amino acid sequence to the avian 100-kDa lysosomal membrane protein LEP100 [Fambrough, D. M., Takeyasu, K., Lippincott-Schwartz, J., Siegel, N. R. & Somerville, D. (1988) J. Cell Biol. 106, 61-67], and to a distinct 110-kDa lysosomal membrane protein (lgp110) that colocalizes with lgp120. The similarities between lysosomal membrane glycoproteins from diverse species, coupled with the fact that at least two distinct lysosomal membrane glycoproteins are expressed in a single species, indicate the existence of a conserved family of glycoproteins enriched in the lysosomal membrane.

Amino Acid Sequence↗

PubMed1983

The anticarcinogenic and tumor growth inhibitory activities of lymphotoxin are associated with altered membrane glycoprotein synthesis.

Membrane glycoprotein synthesis was studied in non-tumorigenic and tumor cells treated with the lymphokine hormone, lymphotoxin, to define the biochemical mechanisms by which lymphotoxin prevents carcinogenesis and inhibits growth of tumor cells. Syrian hamster lymphotoxin increased the synthesis of high molecular weight (50,000-250,000) membrane glycoproteins in non-tumorigenic secondary passage NIH/N Syrian hamster embryo fibroblasts and caused a decrease in the synthesis of high molecular weight glycoproteins in benzo[a]pyrene-induced hamster tumor cells. Increased glycoprotein synthesis varied directly with lymphotoxin concentration and duration of treatment and occurred contemporaneously with lymphotoxin-initiated prevention of carcinogen-induced morphologic transformation. These lymphotoxin-stimulated fluctuations in membrane glycoprotein synthesis are evidence of specific biochemical target cell changes associated with the anticarcinogenic and tumor-inhibitory actions of this immunologic hormone.

Animals↗

PubMed1975

Effect of inhibiting protein synthesis on axonal transport of membrane glycoproteins in an identified neuron of Aplysia.

Intrasonic injection of L-[3H]fucose into R2, the cholinergic giant neuron in the abdominal ganglion of the marine molluse, Aplysia californica, labeled 5 major glycoprotein membrane components, 3 of which were preferentially exported into the axon. Brief exposure to anisomycin, a potent inhibitor of protein synthesis in Aplysia, almost completely blocked the appearance of [3H]glycoprotein in the axon; but fucosylation of proteins in the cell body was only partially inhibited. Polyacrylamide gel electrophoresis showed that the 5 normal components were present in the cell body of inhibited neurons. Thus, synthesis and insertion of glycoproteins into membranes do not guarantee export from the cell body: presumably completion of transportable organelles requires continuous synthesis of new proteins. Longer exposure before injection resulted in reduced amounts of four of the glycoproteins, but one component continued to be formed. Anisomycin can be used to resolve export of glycoproteins out of the cell body from subsequent movement along the axon. When we exposed R2 to anisomycin after injection, [3H]glycoproteins, which had already entered the axon, were translocated along the axon normally; they were distributed in waves, the most distal moving at 50-60 mm/day.

Acetylcholine↗

PubMed1994

The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein.

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60-kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.

Animals↗

PubMed1988

Purification of the 170- to 180-kilodalton membrane glycoprotein associated with multidrug resistance. 170- to 180-kilodalton membrane glycoprotein is an ATPase.

170-180-kDa membrane glycoprotein (P-glycoprotein) associated with multidrug resistance is involved in drug transport mechanisms across the plasma membrane of resistant cells. From sequence analysis of cDNAs of the P-glycoprotein gene, it is postulated that the active drug-efflux pump function may be attributable to the protein. However, purification of the P-glycoprotein while preserving its enzymatic activity has not been reported. In this study, we have purified the P-glycoprotein from the human myelogenous leukemia K562 cell line resistant to adriamycin (K562/ADM) by means of one-step immunoaffinity chromatography using a monoclonal antibody against P-glycoprotein. The procedure was simple and efficiently yielded an electrophoretically homogeneous P-glycoprotein sample. By solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the purified P-glycoprotein was found to have ATPase activity. This ATP hydrolysis may be coupled with the active efflux of anticancer drugs across the plasma membrane of multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1980

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins.

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.

Alkaline Phosphatase↗

PubMed1978

In vitro synthesis of vesicular stomatitis virus membrane glycoprotein and insertion into membranes.

Translation in vitro of the mRNA coding for the vesicular stomatitis virus membrane glycoprotein G in a membrane-free ribosomal extract from HeLa cells allowed the synthesis of only the unglycosylated protein G1 (molecular weight, 63,000). Addition of stripped crude microsomal membranes from HeLa cells resulted in the conversion of G1 to the glycosylated protein G2 (molecular weight, 67,000). The G2 protein synthesized by the reconstructed microsomal membrane/ribosome system was found to be segregated inside the microsomal membrane vesicles and was thus protected from the proteolytic action of trypsin and chymotrypsin. Stripped membranes were required at an early stage of protein synthesis for the synthesized protein to be inserted into the membrane vesicles and to be glycosilated. The segregated protein G2, however, was not completely protected from proteolytic digestion, showing that a portion of the polypeptide chain of about 3000 daltons was present on the cytoplasmic side of the membrane vesicle. Our data thus suggest that, unlike the secretory proteins, the membrane glycoproteins are not completely discharged across the microsomal membranes.

Chymotrypsin↗

PubMed1980

Biogenesis of plasma membrane glycoproteins. Tracer kinetic study of two rat liver plasma membrane glycoproteins in vivo.

Antibodies to purified nucleotide pyrophosphatase (NPPase) and dipeptidyl peptidase IV (DPP IV) were used to study the biogenesis of these rat liver plasma membrane glycoproteins in vivo. Following injection of tritiated leucine, the radioactivity in NPPase and DPP IV decayed at markedly different rates in the plasma membrane, with apparent half-lives of about 1 and 5 days, respectively. In short term experiments, labeling of total plasma membrane proteins was rapid and insensitive to colchicine, while labeling of both NPPase and DPP IV showed a lag of about 15 min, followed by colchcine-sensitive/cycloheximide-insensitive increases to half-maximal and maximal values at about 1 and 2 h, respectively. A peak of labeled DPP IV in rough microsomes at 15 min showed increased mobility on polyacrylamide gels and was largely inaccessible to antibodies in intact microsomes, consistent with its being an underglycosylated precursor, exposed on the cisternal side of the rough endoplasmic reticulum. In contrast, the behavior of unlabeled DPP IV in preparations of rough microsomes and Golgi was consistent with its being contributed by contaminating right-side-out plasma membrane vesicles. This conclusion was also necessary to fit the tracer kinetic data to a simple membrane-flow model, which gave precursor pools (1 microgram/g of liver) and fluxes (1 microgram/h/g of liver) for both DPP IV and NPPase which were about 3 orders of magnitude less than those for the synthesis of rat serum albumin. Thus, unlike hepatoma tissue culture cells (Doyle, D., Baumann, H., England, B., Friedman, E., Hou, E., and Tweto, J. (1978) J. Biol. Chem. 253, 967-973), normal rat liver does not contain large amounts of preformed intracellular plasma membrane precursors.

Animals↗