Identification and subcellular distribution of adipocyte peptides and phosphopeptides.

Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.     

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein: Is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca²⁺ signalling and maintenance of Ca²⁺ homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca²⁺-ATPase, Na⁺, K⁺-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca²⁺ ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca²⁺ entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca²⁺ entry, and their formation and rebuilding have an important regulatory role in cellular Ca²⁺ homeostasis.     

Insulin degradation. XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy.

The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit gamma-globulin was examined by electron microscopy. In cells with intact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.     

Insulin degradation XXIII. Distribution of glutathione-insulin transhydrogenase in isolated rat hepatocytes as studied by immuno-ferritin and electron microscopy

The distribution of glutathione-insulin transhydrogenase (glutathione: protein-disulphide oxidoreductase, EC 1.8.4.2) in isolated rat hepatocytes that had been first treated with rabbit antiserum against purified rat liver transhydrogenase and then with ferritin-conjugated goat anti-rabbit γ-globulin was examined by electron microscopy. In cells with antact plasma membrane, the immunoferritin labeling of glutathione-insulin transhydrogenase was observed on a few external microvillous projections at the outside of the cell. In cells with breaks in the plasma membrane, the immunoferritin labeling appeared extensively on smooth vesicles just inside the plasma membrane and on smooth endoplasmic reticulum extending to and including the outer nuclear membrane, in addition to the external microvillous projections. There was some immunoferritin labeling on rough endoplasmic reticulum and on the inner surface of the plasma membrane. The mitochondria and the outer surface of the plasma membrane of the cell did not show the ferritin labeling. Control parallel samples in which the antiserum was substituted with normal (i.e. non-immune) serum or with neutralized antiserum (prepared by absorption with the transhydrogenase) showed little or no immunoferritin labeling. These results are consistent with the idea that gluthalione-insulin transhydrogenase probably synthesized in the endoplasmic reticulum and that the transhydrogenase accessible to cell surface (or found in the isolated plasma membrane preparations) probably represents a functional continuity between the endoplasmic reticulum and the plasma membrane.     

Cell-Free Transfer of Phosphatidylinositol between Membrane Fractions Isolated from Soybean

Transfer of phosphatidylinositol (PI) between membranes was reconstituted in a cell-free system using membrane fractions isolated from dark-grown soybean (Glycine max [L.] Merr.). Donor membrane vesicles contained [3H]myo-inositol-labeled PI. A fraction enriched in endoplasmic reticulum was a more efficient donor than its parent microsomal membrane fraction. As acceptor, cytoplasmic side-out plasma membrane vesicles were more efficient than cytoplasmic side-in plasma membrane vesicles. Endoplasmic reticulum was also an efficient acceptor, suggesting that transfer occurred to cytoplasmic membrane leaflets. PI transfer was time and temperature dependent but did not require cytosolic proteins, ATP, GTP, cytosol, and acyl-coenzyme A. These results suggest that neither lipid transfer proteins nor transition vesicles, similar to those involved in vesicle trafficking from endoplasmic reticulum to the Golgi apparatus, were involved. In the presence of Mg2+ and ATP, endoplasmic reticulum PI was not metabolized, whereas PI transferred to the plasma membrane was metabolized into phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate. To summarize, the cell-free transfer of endoplasmic reticulum-derived PI was distinct from, for example, vesicle transport from endoplasmic reticulum to Golgi apparatus, not only in its regulation but also in its acceptor unspecificity.     

Dengue virus-induced modifications of host cell membranes.

Enzymatic markers and electron microscopy were utilized to determine the cellular origin of the membrane types isolated from type 2 dengue virus-infected BHK cells by discontinuous sucrose gradient centrifugation. The results showed an apparent separation of plasma membrane, smooth and rough endoplasmic reticulum with increasing density. Virus-induced protein and RNA synthesis, as indicated by the incorporation of radiolabled precursors, was localized on the rough endoplasmic reticulum. Glycosylation, measured by the incorporation of radiolabeled glucosamine into membrane-associated proteins, was most active in the bands of intermediate and smooth endoplasmic reticulum. Polyacrylamide gel electrophoresis of isolated membrane bands, radiolabeled in the presence of actinomycin D, after pulse inhibition by cycloheximide, revealed seven virus-specific proteins associated with all membrane fractions. Viral structural protein V-3, and nonstructural proteins NV-3 and NV-2, increased with decreasing density, whereas NV-5 and NV-4 remained constant. The viral capsid protein V-2 was depleted in the intermediate and smooth endoplasmic reticulum, suggesting that these membranes may serve as the sites for viral maturation. NV-3 was the most prominent virus-specified protein found in the plasma membrane.     

Ca-pumps in smooth muscle: one in plasma membrane and another in endoplasmic reticulum

For several years it has been debated whether the Ca-pump in smooth muscle is located in the plasma membrane or in the endoplasmic reticulum (alias sarcoplasmic reticulum). Experimental evidence using skinned smooth muscle cells and subcellular membrane fractions isolated from a number of smooth muscles is reviewed here to hopefully resolve this issue. The inescapable conclusion is that there are two modes of nonmitochondrial ATP-dependent Ca-transport. The first one, unaffected by oxalate, is localized in the plasma membranes and the second, potentiated by oxalate, is localized in the endoplasmic reticulum. Clear experiments to delineate the roles of the two pumps in the excitation-contraction cycle of the smooth muscle remain to be conducted.     

Budding of Rous sarcoma virus and vesicular stomatitis virus from localized lipid regions in the plasma membrane of chicken embryo fibroblasts

The origin of the envelope lipids acquired by Rous sarcoma virus (RSV) and vesicular stomatitis virus (VSV) during budding from the plasma membrane of chicken embryo fibroblasts was examined. Several differences were observed between the lipid composition of RSV and the plasma membrane. When the phospholipid composition of the cells was modified by growing them in the presence of the choline analogues, N,N-dimethylethanolamine or l-2-amino-1-butanol, the phospholipid composition of the virus was subsequently altered but in a very different manner than the plasma membrane. In the plasma membrane, the increase in the analogue-containing phospholipid was at the expense of phosphatidylcholine and phosphatidylethanolamine while the amount of sphingomyelin remained constant. In RSV, however, there was a decrease in sphingomyelin and phosphatidylethanolamine while there was only a small change in the amount of phosphatidylcholine. Phospholipid polar head group modification did not significantly alter the fatty acid composition or the cholesterol content. Membranes of phagosomes isolated after the cells had ingested latex beads had essentially the same phospholipid composition as the plasma membrane. The phospholipid composition of VSV was different from RSV, but it also did not reflect the composition of the plasma membrane. The composition of the plasma membrane was intermediate between the viruses and the endoplasmic reticulum, but contamination of the plasma membrane fraction with the endoplasmic reticulum could not account for the observed differences. These results show that the viruses bud from localized lipid regions that do not reflect the average properties of the plasma membrane.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Subcellular distribution and regulation of hepatic bilirubin UDP-glucuronyltransferase

We have investigated the subcellular location and regulation of hepatic bilirubin UDP-glucuronyltransferase, which has been presumed to be located largely in the smooth endoplasmic reticulum. Purity of subcellular membrane fractions isolated from rat liver was assessed by electron microscopy and marker enzymes. Bilirubin UDP-glucuronyltransferase activity was measured by radiochemical assay using a physiologic concentration of [14C]bilirubin, and formation rates of bilirubin diglucuronide and monoglucuronides (C-8 and C-12 isomers) were determined. Activity of the enzyme was widely distributed in subcellular membranes, the majority being found in smooth and rough endoplasmic reticulum, with small amounts in nuclear envelope and Golgi membranes. No measurable activity was found in plasma membranes or in cytosol. Synthesis of bilirubin diglucuronide as a percentage of total conjugates and the ratio of C-8/C-12 bilirubin monoglucuronide isomers formed were comparable in all membranes, suggesting that the same enzyme is present in all locations. However, the regulation of bilirubin UDP-glucuronyltransferase activity differed among intracellular membranes; enzyme activity measured in the presence of the allosteric effector uridine 5'-diphospho-N-acetylglucosamine exhibited latency in smooth endoplasmic reticulum and Golgi membranes, but not in rough endoplasmic reticulum and nuclear envelope. Since rough membranes comprise 60% of hepatocyte endoplasmic reticulum and bilirubin UDP-glucuronyltransferase activity in vitro is maximal in this membrane fraction under presumed physiologic conditions, it is likely that the rough endoplasmic reticulum represents the major site of bilirubin glucuronidation in hepatocytes.     

A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.     

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes

Summary A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A⁺ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.     

Subcellular distribution and biosynthesis of rat liver gangliosides

Gangliosides have generally been assumed to be localized primarily in the plasma membrane. Analysis of gangliosides from isolated subcellular membrane fractions of rat liver indicated that 76% of the total ganglioside sialic acid was present in the plasma membrane. Mitochondria and endoplasmic reticulum fractions, while containing only low levels of gangliosides on a protein basis, each contained approx. 10% of total ganglioside sialic acid. Gangliosides also were present in the Golgi apparatus and nuclear membrane fractions, and soluble gangliosides were in the supernatant. Individual gangliosides were non-homogeneously distributed and each membrane fraction was characterized by a unique ganglioside composition. Plasma membrane contained only 14 and 28% of the total GD1a and GD3, respectively, but 80-90% of the GM1, GD1b, GT1b and GQ1b. Endoplasmic reticulum, when corrected for plasma membrane contamination, contained only trace amounts of GM1, GD1b, GT1b and GQ1b, but 11 and 5% of the total GD1a and GD3, respectively. The ganglioside composition of highly purified endoplasmic reticulum was similar. Ganglioside biosynthetic enzymes were concentrated in the Golgi apparatus. However, low levels of these enzymes were present in the highly purified endoplasmic reticulum fractions. Pulse-chase experiments with [3H]galactose revealed that total gangliosides were labeled first in the Golgi apparatus, mitochondria and supernatant within 10 min. Labeled gangliosides were next observed at 30 min in the endoplasmic reticulum, plasma membrane and nuclear membrane fractions. Analysis of the individual gangliosides also revealed that GM3, GM1, GD1a and GD1b were labeled first in the Golgi apparatus at 10 min. These studies indicate that gangliosides synthesized in the Golgi apparatus may be transported not only to the plasma membrane, but to the endoplasmic reticulum and to other internal endomembranes as well.     

Passage of viral membrane proteins through the Golgi complex

BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes.     

Isolation of plasma membrane,Golgi apparatus,and endoplasmic reticulum fractions from single homogenates of mouse liver

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose-6-phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-cytochrome c reductase and contain membrane vesicles with attached ribosomes. K⁺-stimulated p-nitrophenyl phosphatase and (Na⁺ K⁺) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.     

The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.     

A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes

In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.     

Alterations in intracellular calcium compartmentation following inhibition of calcium efflux from isolated hepatocytes

Addition of ATP to the incubation medium of freshly isolated rat hepatocytes causes a marked inhibition of the efflux of Ca2+ from the cells, and its accumulation in intracellular compartments. After an initial rise in cytosolic free Ca2+ concentration, as indicated by the activation of phosphorylase, Ca2+ is preferentially sequestered in the mitochondria, without any apparent contribution by the endoplasmic reticulum. Impairment of mitochondrial Ca2+ homeostasis by pyridine nucleotide oxidation associated with tert-butyl hydroperoxide metabolism, prevents the ATP-dependent cellular Ca2+ accumulation and causes a release of Ca2+ from the hepatocytes into the medium. Conversely, maintenance of the mitochondrial pyridine nucleotides in a more reduced state, e. g. in presence of 3-hydroxybutyrate in the medium, prevents this hydroperoxide-induced release of intracellular Ca2+. Under conditions of impaired mitochondrial Ca2+ sequestration, there appears to be a redistribution of a minor fraction of the intracellular Ca2+ from the mitochondria to the endoplasmic reticulum. Our results provide additional evidence for the critical involvement of the plasma membrane Ca2+-extruding system in the physiological regulation of the cytosolic free Ca2+ concentration in hepatocytes, and suggest that the mitochondria play a more important role than the endoplasmic reticulum in the regulation of the cytosolic free Ca2+ level when the plasma membrane Ca2+ pump is inhibited.     

Distribution of insulin receptors among mouse liver endomembranes.

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. 125I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10--20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1--2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0-10(8) M-1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na+ + K+ + Mg2+)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.     

Subcellular localization of phospholipids and enzymes involved in PAF-acether metabolism

The biosynthesis of platelet-activating factor (PAF-acether or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) through the remodeling pathway was investigated at the subcellular level in two different cell lines. In human neutrophils, plasma membrane was isolated not only from granules, but also from internal membranes related to endoplasmic reticulum. Interestingly, the latter exhibited enhanced acetyltransferase upon neutrophil stimulation with ionophore A23187. A similar study was undertaken on the tumor strain Krebs-II cells. The enzyme acetyltransferase was found to be located only on an endoplasmic reticulum subfraction, whereas most alkylacyl-GPC, the source of PAF-precursor alkyl-lyso-GPC, was located in the plasma membrane inner leaflet. The topographical separation of enzyme and precursor emphasizes the central role of the intracellular phospholipase A2 in providing lyso-PAF to the acetyltransferase to form PAF-acether.