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.     

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

The size and detergent binding of membrane proteins.

Sucrose density gradient centrifugation has been used to measure the binding of Triton X-100 above its critical micellar concentration to a variety of purified membrane and non-membrane proteins. In addition, binding studies were done on the three proteins below the critical micellar concentration of detergent to distinguish between the interaction of proteins with detergent monomers and detergent micelles. A procedure is described for the calculation of the molecular weight of these Triton X-100 protein complexes and measurements were made for opsin, plasma low density lipoprotein, the (Na-+ plus K-+)-dependent adenosine triphosphatase, the human red blood cell major sialoglycoprotein (PAS-1) and the human red blood cell minor glycoprotein (bandIII). These proteins behave as monomers or dimers in detergent and bind between 0.28 and 1.12 g of detergent per g of protein. A general method is also present for calculating the molecular size and shape of impure membrane proteins in detergent. Finally, Triton X-100 was shown to replace bound Na dodecyl-SO4 on the minor glycoprotein of the red blood cell.     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver plasma membrane

Phosphatidylethanolamine:ceramide-ethanolaminephosphotransferase catalyzes the synthesis of ceramide-ethanolamine, a sphingomyelin analogue. Its transverse localization in rat liver plasma membrane was studied by treating intact and deoxycholate- or Triton X-100-disrupted membrane vesicles with trypsin or bacterial protease. The latency of ATPase was preserved during protease treatment; its value was 80% in the membrane vesicles obtained by sucrose gradient procedure alone and 91.2% in the vesicles isolated after sucrose gradient plus two-phase partitioning. This suggested that membrane integrity was not altered and that 90% of the vesicles were right-side out. When the sucrose gradient was followed by the two-phase procedure, 62% of phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase was accessible to the protease action, but only 45% in vesicles obtained by sucrose gradient alone. Our results suggest that at least a sizable portion of the active center of the enzyme responsible of biosynthesis of ceramide-phosphoethanolamine is located on the external side of liver plasma membrane and that the other is embedded in the membrane interior and is not accessible to trypsin, even in the presence of detergent.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.     

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.     

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.     

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 μg of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio.The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radioactivity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel .     

Solubilization of the membrane bound lactose specific component of the staphylococcal PEP dependant phosphotransferase system

The membrane bound lactose specific component of the PEP dependant phosphotransferase system of Staphylococcus aureus has been solubilized using the non ionic detergent Triton X-100. Some properties of the crude soluble enzyme are reported.     

Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

A cytoskeleton-associated plasma membrane heparan sulfate proteoglycan in Schwann cells

Schwann cells cocultured with sensory neurons in a serum-free medium accumulate a single species of radiolabeled heparan sulfate proteoglycan (HS-PG) during incubation in medium containing 35SO4. This HS-PG was poorly extracted from cultures by solutions containing 1% Triton X-100 in low salt buffer or by solutions containing 1 M KCl, 4 M urea plus dithiothreitol, 1 mM Tris-HCl, 5 mM EDTA, or 100 micrograms/ml of heparin. The HS-PG was efficiently extracted, however, by 1% Triton X-100 in the presence of 1 M KCl or by 1% deoxycholate. These treatments solubilize both cell membranes and the Schwann cell cytoskeleton. In intact cells the HS-PG was digested by trypsin, indicating it was at least partially exposed on the cell surface. When solubilized HS-PG was applied to a column of octyl-sepharose CL-4B, more than 90% was retained by the column, but was quantitatively eluted by a solution containing 1% Triton X-100. In addition, the solubilized HS-PG could be incorporated into artificial phospholipid vesicles. These results indicate the HS-PG is an integral plasma membrane protein. The inability of low ionic strength solutions containing Triton X-100 to solubilize the HS-PG suggested it was bound to an additional structure. To determine whether the HS-PG was associated with the cytoskeleton we isolated cytoskeletons by detergent lysis of cells and centrifugation. The major protein components of isolated cytoskeletons were spectrin (Mr 225,000), vimentin (Mr 58,000), and actin (Mr 45,000). When 35SO4-labeled cells were used to prepare cytoskeletons approximately 80% of the total HS-PG was recovered in the cytoskeleton fraction. These results suggest the HS-PG is an externally exposed integral plasma membrane protein that is anchored to the Schwann cell cytoskeleton.     

Solubilization of human erythrocyte membrane glycoproteins by triton X-100.

1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.     

Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane

Membrane cholesterol-sphingolipid 'rafts', which are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold, have been implicated in the sorting of certain membrane proteins, such as placental alkaline phosphatase (PLAP), to the apical plasma membrane domain of epithelial cells. Here we show that prominin, an apically sorted pentaspan membrane protein, becomes associated in the trans-Golgi network with a lipid raft that is soluble in Triton X-100 but insoluble in another non-ionic detergent, Lubrol WX. At the cell surface, prominin remains insoluble in Lubrol WX and is selectively associated with microvilli, being largely segregated from the membrane subdomains containing PLAP. Cholesterol depletion results in the loss of prominin's microvillus-specific localization but does not lead to its complete intermixing with PLAP. We propose the coexistence within a membrane domain, such as the apical plasma membrane, of different cholesterol-based lipid rafts, which underlie the generation and maintenance of membrane subdomains.     

Evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes

The cellular distribution of carbonic anhydrase is a key characteristic for the role of the enzyme in cell function. In several epithelia involved in bicarbonate transport this enzyme is located in the plasma membrane. Because bicarbonate secretion is an important mechanism in bile formation by the liver, we investigated the presence of carbonic anhydrase activity in isolated plasma membranes from rat hepatocytes. Carbonic anhydrase activity was enriched 1.79-fold in plasma membrane preparations. This activity was inhibited by acetazolamide and activated by Triton X-100, but was insensitive to Cl- or CNO-. It is highly unlikely that the low contamination of cytoplasm and intracellular membranes could account for the presence of carbonic anhydrase activity in plasma membrane preparations. Moreover, the results from resuspension/washing of plasma membrane fractions in ionic media suggest an absence of soluble carbonic anhydrase adsorption upon plasma membrane. Accordingly, the present findings provide strong evidence for the presence of carbonic anhydrase in the plasma membrane of rat hepatocytes.     

Evidence for a synaptic plasma membrane associated adenylate kinase in the rat brain

About 5% of the total adenylate kinase activity in the rat forebrain was found in a subcellular fraction enriched in synaptic plasma membrane (SPM). The enzyme remained membrane bound after washing by 1M potassium acetate. It was resistant to trypsin digestion under conditions which destroyed 90% of acetylcholinesterase activity. The SPM enzyme was solubilized by 0.25% Triton X-100 resulting in a 4-fold increase in activity. Similar effects were observed when SPM was treated with phospholipases, melittin and trifluoperazine. These results suggest the occurrence of an adenylate kinase closely associated with SPM the activity of which can only be fully expressed by disturbances to the hydrophobic lipid bilayer. The enzyme can be seen as strategically located to play a role in regenerating ATP required for the manifold activities of the synaptic membrane.     

Triton X-100 solubilization of mitochondrial inner and outer membranes

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-micro-scopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzymeQ) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane enzyme activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochromec reductase activity practically disappears even at low Triton X-100 concentrations.     

Structure of human platelet membrane glycoproteins IIb and IIIa as determined by electron microscopy

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes and examined for glycoprotein stoichiometry and morphology. To determine the ratio of glycoproteins in the complex, the isolated glycoproteins were solubilized with sodium dodecyl sulfate and separated by high-performance liquid chromatography. Quantitative amino acid analysis of individual glycoproteins showed that the ratio of GP IIb to GP IIIa in the Ca2+-dependent complex was 0.93:1. Morphology was determined by electron microscopy of rotary-shadowed and negatively stained specimens. Individual complexes consisted of two domains: an oblong head of approximately 8 X 10 nm with two rodlike tails extending approximately 14-17 nm from one side of the head. Treatment of the isolated complex with EDTA resulted in the appearance of a mixture of oblong and filamentous structures, which could be separated by a sucrose gradient sedimentation in Triton X-100. As seen by rotary and unidirectional shadowing, GP IIb was a compact structure, approximately 8 X 10 nm in size. Isolated GP IIIa was more heterogeneous but was most often observed in an elongated form, varying in length from 20 to 30 nm and in width from 2 to 3 nm. By comparing these structures to that of the heterodimer complex, it was determined that the oblong domain was GP IIb and the rodlike tails were GP IIIa. Each milligram of isolated GP IIb-IIIa complex bound 0.30 mg of [3H]Triton X-100, indicating that the glycoprotein complex contained limited hydrophobic domains. Upon removal of detergent, GP IIb-IIIa complexes formed aggregates that sedimented in sucrose gradients as a diffuse peak ranging from 14 to 32 s. Examination of these aggregates by electron microscopy showed that they were composed of clusters or "rosettes" of 2 to 20 or more of the GP IIb-IIIa complexes. The orientation of these rosettes was such that the tails were joined in the center, with the head portions directed away from the interacting tails. It thus appears that the primary hydrophobic domains of the GP IIb-IIIa complex exist at the tips of the GP IIIa tails. Because the GP IIb-IIIa complex is an intrinsic membrane glycoprotein, these findings indicate a potential membrane attachment site for the GP IIb-IIIa complexes.