Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of d-galactosamine

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

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PROTEINS OF THE POSTSYNAPTIC DENSITY

The Journal of Cell Biology ◽

10.1083/jcb.63.2.456 ◽

1974 ◽

Vol 63 (2) ◽

pp. 456-465 ◽

Cited By ~ 76

Author(s):

G. Banker ◽

L. Churchill ◽

C. W. Cotman

Keyword(s):

Molecular Weight ◽

Plasma Membrane ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Composition ◽

Synaptic Plasma Membrane ◽

Brain Protein ◽

Single Polypeptide ◽

Structural Matrix ◽

Polypeptide Band

An analysis was made of the protein composition of a fraction of postsynaptic densities (PSDs) prepared from rat brain. Protein makes up 90% of the material in the PSD fraction. Two major polypeptide fractions are present, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The major polypeptide fraction has a molecular weight of 53,000, makes up about 45% of the PSD protein, and comigrates on gels with a major polypeptide of the synaptic plasma membrane. The other polypeptide band has a molecular weight of 97,000, accounts for 17% of the PSD protein, and is not a prominent constituent of other fractions. Six other polypeptides of higher molecular weight (100,000–180,000) are consistently present in small amounts (3–9% each). The PSD fraction contains slightly greater amounts of polar amino acids and proline than the synaptic plasma membrane fraction, but no amino acid is usually prominent. The PSD apparently consists of a structural matrix formed primarily by a single polypeptide or class of polypeptides of 53,000 molecular weight. Small amounts of other specialized proteins are contained within this matrix.

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Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini.

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5541 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5541-5550 ◽

Cited By ~ 140

Author(s):

J R Grove ◽

P Banerjee ◽

A Balasubramanyam ◽

P J Coffer ◽

D J Price ◽

...

Keyword(s):

Rat Liver ◽

Kinase Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cloning And Expression ◽

S6 Kinase ◽

P70 S6 Kinase ◽

Cos Cells ◽

Sds Page

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

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Downregulation of surface sodium pumps by endocytosis during meiotic maturation of Xenopus laevis oocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1990.258.1.c179 ◽

1990 ◽

Vol 258 (1) ◽

pp. C179-C184 ◽

Cited By ~ 59

Author(s):

G. Schmalzing ◽

P. Eckard ◽

S. Kroner ◽

H. Passow

Keyword(s):

Plasma Membrane ◽

Xenopus Laevis ◽

Sucrose Gradient ◽

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Buoyant Density ◽

Sodium Dodecyl ◽

Meiotic Maturation ◽

Xenopus Laevis Oocytes ◽

Sodium Pumps

During meiotic maturation, plasma membranes of Xenopus laevis oocytes completely lose the capacity to transport Na and K and to bind ouabain. To explore whether the downregulation might be due to an internalization of the sodium pump molecules, the intracellular binding of ouabain was determined. Selective permeabilization of the plasma membrane of mature oocytes (eggs) by digitonin almost failed to disclose ouabain binding sites. However, when the eggs were additionally treated with 0.02% sodium dodecyl sulfate (SDS) to permeabilize inner membranes, all sodium pumps present before maturation were recovered. Phosphorylation by [gamma-32P]ATP combined with SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography showed that sodium pumps were greatly reduced in isolated plasma membranes of eggs. According to sucrose gradient fractionation, maturation induced a shift of sodium pumps from the plasma membrane fraction to membranes of lower buoyant density with a protein composition different from that of the plasma membrane. Endocytosed sodium pumps identified on the sucrose gradient from [3H]ouabain bound to the cell surface before maturation could be phosphorylated with inorganic [32P]phosphate. The findings suggest that downregulation of sodium pumps during maturation is brought about by translocation of surface sodium pumps to an intracellular compartment, presumably endosomes. This contrasts the mechanism of downregulation of Na-dependent cotransport systems, the activities of which are reduced as a consequence of a maturation-induced depolarization of the membrane without a removal of the corresponding transporter from the plasma membrane.

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Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

Biochemical Journal ◽

10.1042/bj2190301 ◽

1984 ◽

Vol 219 (1) ◽

pp. 301-308 ◽

Cited By ~ 51

Author(s):

A A Davies ◽

N M Wigglesworth ◽

D Allan ◽

R J Owens ◽

M J Crumpton

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Specific Activity ◽

Gradient Centrifugation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Fold Increase ◽

Insoluble Fraction ◽

Insoluble Residue ◽

Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

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Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5541-5550.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5541-5550

Author(s):

J R Grove ◽

P Banerjee ◽

A Balasubramanyam ◽

P J Coffer ◽

D J Price ◽

...

Keyword(s):

Rat Liver ◽

Kinase Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cloning And Expression ◽

S6 Kinase ◽

P70 S6 Kinase ◽

Cos Cells ◽

Sds Page

Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5' region, such that the longer polypeptide (p70 S6 kinase alpha I; calculated Mr 58,946) consists of 525 amino acids, of which the last 502 residues are identical in sequence to the entire polypeptides encoded by the second cDNA (p70 S6 kinase alpha II; calculated Mr 56,153). Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase alpha I cDNA through the utilization of different translational start sites. Although the two p70 S6 kinase polypeptides differ by only 23 amino acid residues, the slightly longer alpha I polypeptide exhibits anomalously slow mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), migrating at an apparent Mr of 90,000 probably because of the presence of six consecutive Arg residues immediately following the initiator methionine. Transient expression of p70 alpha I and alpha II S6 kinase cDNA in COS cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Upon immunoblotting, the recombinant p70 polypeptides appear as a closely spaced ladder of four to five bands between 65 and 70 kDa (alpha II) and 85 and 90 kDa (alpha I). Transfection with the alpha II cDNA yields only the smaller set of bands, while transfection with the alpha I cDNA generates both sets of bands. Mutation of Met-24 in the alpha I cDNA to Leu or Thr suppresses synthesis of the alpha II polypeptides. Only the p70 alpha I and alpha II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70- and 90-kDa proteins observed in purified rat liver S6 kinase. Moreover, it is the recombinant p70 polypeptides of slowest mobility that coelute with S6 kinase activity on anion-exchange chromatography. The slower mobility and higher enzymatic activity of these p70 proteins is due to Ser/Thr phosphorylation, inasmuch as treatment with phosphatase 2A inactivates kinase activity and increases the mobility of the bands on SDS-PAGE in an okadaic acid-sensitive manner. Thus, the recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

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Role of membrane-bound and free polyribosomes in the synthesis of cytochrome c in rat liver

Biochemical Journal ◽

10.1042/bj1400157 ◽

1974 ◽

Vol 140 (2) ◽

pp. 157-167 ◽

Cited By ~ 12

Author(s):

Néstor F. González-Cadavid ◽

Carmen Sáez De Córdova

Keyword(s):

Endoplasmic Reticulum ◽

Cytochrome C ◽

Rat Liver ◽

Cell Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Mitochondrial Protein ◽

Sodium Dodecyl ◽

Membrane Bound

The functional distinction of membrane-bound and free polyribosomes for the synthesis of exportable and non-exportable proteins respectively is not so strict as was initially thought, and it was therefore decided to investigate their relative contribution to the elaboration of an internal protein integrated into a cell structure. Cytochrome c was chosen as an example of a soluble mitochondrial protein, and the incorporation of [14C]leucine and δ-amino[14C]laevulinate into the molecule was studied by using different ribosomal preparations from regenerating rat liver. A new procedure was devised for the purification of cytochrome c, based on ion-exchange chromatography combined with sodium dodecyl sulphate–polyacrylamide-gel electrophoresis. In spite of cytochrome c being a non-exportable protein, the membrane-bound polyribosomes were at least as active as the free ribosomes in the synthesis in vitro of the apoprotein and the haem moiety. The detergent-treated ribosomes could also effect the synthesis of cytochrome c, although at a lower rate. Since in liver more than two-thirds of the ribosomes are bound to the endoplasmic-reticulum membranes, it is considered that in vivo they are responsible for the synthesis of most of the cytochrome c content of the cell. This suggests that in secretory tissues the endoplasmic reticulum plays a predominant role in mitochondrial biogenesis, although free ribosomes may participate in the partial turnover of some parts of the organelle. The hypothesis on the functional specialization of the different kinds of ribosomes was therefore modified to account for their parallel intervention in the synthesis of proteins associated with membranous structures.

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Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.5.g894 ◽

1993 ◽

Vol 265 (5) ◽

pp. G894-G902 ◽

Cited By ~ 25

Author(s):

D. D. Stump ◽

S. L. Zhou ◽

P. D. Berk

Keyword(s):

Plasma Membrane ◽

Fatty Acid ◽

Amino Acid ◽

Rat Liver ◽

Aspartate Aminotransferase ◽

High Performance ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Fatty Acid Binding ◽

Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

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D-Glucose uptake and insulin binding by the human adipose cell plasma membrane as a function of its polypeptide composition

Canadian Journal of Biochemistry ◽

10.1139/o77-020 ◽

1977 ◽

Vol 55 (2) ◽

pp. 126-133 ◽

Cited By ~ 5

Author(s):

Bluma G. Brenner ◽

Shiro Ozaki ◽

Norman Kalant ◽

Arthur Kahlenberg

Keyword(s):

Plasma Membrane ◽

Glucose Uptake ◽

Plasma Membranes ◽

Binding Capacity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Insulin Binding ◽

Partial Purification ◽

Scatchard Analysis ◽

Molecular Weight Range

A preparation of plasma membranes isolated from human omental lipocytes is composed of about 15 major polypeptide components including three major glycoproteins with an apparent molecular weight range from 100 000 to 23 000, as determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Extraction of this membrane preparation with sodium iodide or 2,3-dimethylmaleic anhydride solubilized 50 and 70% of the membrane protein, respectively, resulting from the extensive extraction of protein from all but two of the major membrane polypeptide components. This removal of protein did not affect the membrane's stereospecific D-glucose-uptake activity but did reduce its total specific [l25I]insulin-binding activity by 46–67%. The binding of [125I]insulin to its specific receptor on lipocyte plasma membranes was detected at physiologic concentrations of the hormone and could be competitively displaced by increasing concentrations of native insulin. The kinetic behaviour of this reaction was approximated by Scatchard analysis, and both the affinity and binding capacity of the plasma membrane for insulin were increased at lower temperatures.These results suggest that D-glucose transport in human adipose tissue is mediated by an intrinsic component of the hydrophobic structure of the lipocyte plasma membrane, and represent a partial purification of this component. In addition, these studies demonstrate and characterize the binding of insulin to the plasma membrane isolated from human lipocytes. A quantitative study of this binding reaction may provide further understanding of the mechanisms underlying the decreased insulin responsiveness characteristic of human diabetes.

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Isolation and electrophoretic characterization of the plasma membrane of sea-urchin sperm

Journal of Cell Science ◽

10.1242/jcs.59.1.13 ◽

1983 ◽

Vol 59 (1) ◽

pp. 13-25

Author(s):

N.L. Cross

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Specific Activity ◽

Periodic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Electrophoretic Patterns ◽

Sperm Plasma Membrane

A subcellular fraction containing plasma membranes was isolated from flagella of the sperm of Strongylocentrotus purpuratus by differential centrifugation, and analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Coomassie Blue staining revealed nine major bands and 14 minor species. Five bands of apparent molecular weights approximately 200 X 10(3), 149 X 10(3), 120 X 10(3), 75 X 10(3) and 59 X 10(3) also stained with periodic acid-Schiff's reagent and so are probably glycoproteins. These five components are externally exposed, as determined by lactoperoxidase-catalysed radio-iodination. Isolation of membranes from radio-iodinated sperm results in an enrichment of about tenfold in the specific activity of 125I. Comparison of the electrophoretic patterns of labelled sperm and of the membranes isolated from 125I-labelled sperm suggests that no major labelled proteins are lost during the isolation procedure, and so to this extent the membrane fraction is representative of the entire sperm plasma membrane.

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