Digestion of insulin receptors with proteolytic and glycosidic enzymes-effects on purified and membrane-associated receptor subunits

Insulin receptors, photoaffinity-labeled in rat liver and human placenta membranes, and receptor purified from rat liver membranes, were treated with proteolytic and glycosidic enzymes and analyzed by SDS-polyacrylamide gel electrophoresis. The protease fragments from the purified receptor were similar to those from the photoaffinity-labeled receptors, if these were solubilized prior to digestion. However, the intact photoaffinity-labeled membranes were less sensitive to proteases, and different fragments were produced. Thus, solubilization exposes sites very sensitive to proteolysis. The labeled tryptic fragments of placenta membranes remained membrane-bound. Neuraminidase altered the mobility of the intact receptor and of several of the large tryptic fragments, indicating that these contain sialic acid and are probably exposed on the outer surface of the membrane.     

Ultrastructural characterization by ruthenium red of the surface of the fat globule membrane of human and rat milk with data on carbohydrates of fractions of rat milk

The fat globules of the cream fractions of human and rat milk were stained with ruthenium red. Under the electron microscope, discrete granules and an amorphous coat of lesser density are seen at the surface of the milk fat globules. Since ruthenium red binds anionic groups selectively, it is probable that the granules contain the greatest concentration of these groups. The cream fraction of rat milk contains hexoses, hexosamines, methylpentoses and sialic acid. Methylpentoses and hexosamines are significantly enriched in the cream fraction. It is concluded that the finding of a surface coat in milk fat globules is in keeping with the Bargmann-Knoop model and suggests a distinct mechanism for carrying certain complex carbohydrates in milk. The role of the negative charges at the outer surface of the membrane coat is maintaining fat globules in suspension and in binding certain cations such as calcium is suggested.     

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

Sialic acid in crude myelin fractions from rat brain.

Protein- and lipid-bound sialic acid was assayed in myelin fractions isolated by four different methods from rat brain homogenates. The extent to which non-myelin cellular membranes contaminate these fractions was assessed by electron microscopy and marker-enzyme assays. Small amounts of sialic acid found in the least contaminated myelin fractions are considered to be constituents of axonal and satellite cell plasma membranes known to be present. The data are discussed with reference to the ultrastructural appearance of myelin.     

The Effect of Isoproterenol upon the Chemical Composition of Plasma Membranes in the Mouse Parotid Gland

A single intraperitoneal injection of isoproterenol induces resting cells from the acini of the mouse parotid gland to enter the proliferative cycle. Parotid plasma membrane from non-stimulated and isoproterenol-treated mice were prepared by differential centrifugation of the homogenates. Comparing the chemical composition of plasma membranes from non-stimulated and isoproterenol-treated mice, no variation in the phospholipid/protein ratio was observed. However, the levels of neutral sugars, hexosamines and sialic acid falls drastically in the early prereplicative phase. The decrease in neutral sugars and hexosamines in plasma membranes caused by isoproterenol is imitated by pilocarpine, which induces secretion but little or no increase in DNA synthesis. However, pilocarpine does not mobilize sialic acid from the plasma membrane. Moreover, dosis of isoproterenol that elicits secretion but not mitosis in the acinar cells, does not induce the movement of sialic acid from the plasma membrane. The mobilization of sialic acid from plasma membranes caused by isoproterenol was also demonstrated in an in vitro system. Treatment of the plasma membrane with chloro-form/methanol shows that around 60% of the sialic acid is present in the less polar phase. We conclude that the separation of sialic acid from the plasma membrane is one of the early steps in the sequence of events leading to DNA synthesis and cell division in the isoproterenol-stimulated parotid gland of mice.     

Gangliosides depleted in plasma membrane are directed to internal membranes of rat hepatomas: evidence for a glycolipid sorting defect in hepatocarcinogenesis

Ganglioside compositions of plasma membrane fractions highly purified from rat liver and hepatomas by phase partitioning were compared with those of fractions composed of internal membranes, free of plasma membrane. With liver, 70-80% of the the lipid bound sialic acid were accounted for by a plasma membrane location. In hepatomas this percentage was reduced to 50-65%. More pronounced was the distribution of the simple monosialoganglioside GM3. In the hepatomas, 60-80% of the GM3 was found associated with internal membranes as compared to liver where only 35% of the GM3 was present in internal membranes. The findings suggest a glycolipid sorting defect in hepatocarcinogenesis where gangliosides, and especially monosialogangliosides, are diverted to internal membranes rather than being correctly transported to the cell surface. Since GM3 is synthesized exclusively in the Golgi apparatus of both liver and hepatomas, the basis for the sorting defect may reside in a functionally altered Golgi apparatus.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

HISTOCHEMICAL STAINING AND CHARACTERIZATION OF GLYCOPROTEINS IN ACID-SECRETING CELLS OF FROG STOMACH

Glycoproteins were histochemically localized in oxyntic cells of the frog stomach by staining with periodic acid-silver methenamine. Reduction of silver was most intense on (a) the outer aspect of the apical plasmalemma, (b) within the tubular smooth membrane system characteristic of oxyntic cells, and (c) within cisternae and vesicles of the Golgi complex. Other membrane components such as those from the mitochondria, nucleus, junctional complex, lateral and basal cell membranes showed little or no stainability. Gastric mucosal homogenates were fractionated by centrifugation for further morphological and chemical analysis. The staining reaction of the microsomal fraction (40,000 g x 60 min) was similar to that of the tubular membranous components of intact oxyntic cells. Carbohydrate analyses showed that all cell fractions are extremely low in acidic sugars, uronic and sialic acids, while neutral sugars and hexosamines are relatively abundant. The microsomal fraction contains the largest proportion of carbohydrates, ca. 9% of the fat-free dry weight. Another distinguishing feature is that glucosamine is the only detectable hexosamine in the microsomal fraction. These histochemical and chemical data indicate that neutral glycoproteins are associated with membranous components which have been implicated in the process of HCl secretion by oxyntic cells. The staining pattern within the cells supports the hypothesis of interrelationships between the Golgi membranes, tubular smooth membranes, and apical surface membrane.     

Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study

When biochemical studies on the liver are interpreted, the cells of the sinusoidal area frequently receive little attention because, compared to hepatocytes, their contribution to subcellular fractions is assumed insignificant. A systematic stereological analysis of liver parenchyma was therefore performed in order to determine the distribution of organelles and membranes between hepatocytic and nonhepatocytic cells, namely endothelial, Kupffer, and fat-storing cells. The livers were fixed by vascular perfusion and the data were corrected for systematic errors dur to section thickness and compression. The extracellular space compartment includes the lumina of sinusoids (10.6%), the space of Disse (4.9%), and the bile canaliculi (0.4%). Hepatocytes constitute 78% of parenchymal volume; the nonhepatocytes account for 6.3% and consist of 2.8% endothelial cells, 2.1% Kupffer cells, and 1.4% fat- storing cells. The nonhepatocytes contribute 55% of the volume of lipid droplets in the liver, 43% of the lysosomes, and 1.2% of the mitochondria. Although the nonhepatocytes account for only 8% of the total surface area of parenchymal membranes, they contain 26.5% of all the plasma membranes, 32.4% of the lysosomal membranes, 15.1% of the Golgi apparatus 6.4% of the endoplasmic reticulum, and 2.4% of the mitochondrial membranes. The data demonstrate the extent to which nonhepatocytic organelles can potentially contaminate subcellular fractions used for biochemical studies. Particularly important for the interpretation of studies on lysosomes, plasma membrane, and Golgi apparatus is the finding that an appreciable part of these organelles may be derived from cell types other than hepatocytes.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

Identification of sialic acids in cell adhesion molecule, contact site A from Dictyostelium discoideum.

It has been believed that Dictyostelium discoideum cell membranes contain no sialic acid. In this study, however, we found that contact site A, the cell adhesion molecule of D. discoideum, is a major glycoprotein containing sialic acids. This suggests that sialic acid in non-reducing terminal plays an important role in the cell adhesion in which contact site A is involved.     

Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.     

Lipid composition of subcellular particles from sheep platelets. Location of phosphatidylethanolamine and phosphatidylserine in plasma membranes and platelet liposomes

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.     

Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes

Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.     

Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

Thymocyte plasma and nuclear membranes obtained by the procedure described in the accompanying paper were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol, sialic aicd; they did not contain nucleic acids. In comparison, nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. 50% of the cellular cholesterol and of the membrane-bound sialic acid were found in the plasma membranes, 14% in the nuclear membranes. Live cells were labeled with 131I, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane market-enzymes was obtained. Label enrichment was about 50-fold in the two lightest of the three plasma membrane fractions. 60% of the label was contained in the plasma membranes, only 4% in the nuclear membranes. Cross-contamination of these two types of membranes was thus negligible. Sodium dodecyl sulfate-gel electrophoresis revealed three different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction, and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high molecular weight glycoproteins could be considered as marker-proteins of, respectively, 280,000, 260,000, and 230,000 daltons. 131I-labeling of live cells tagged with a very high specific activity three glycoproteins of mol wt 280,000, 200,000, and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.     

Muscle surface membranes: preparative methods affect apparent chemical properties and neurotoxin binding.

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050--1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using alpha-[125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p-nitrophenylphosphatase, higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.     

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain.

The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M.     

Association of kinesin with characterized membrane-bounded organelles.

The family of molecular motors known as kinesin has been implicated in the translocation of membrane-bounded organelles along microtubules, but relatively little is known about the interaction of kinesin with organelles. In order to understand these interactions, we have examined the association of kinesin with a variety of organelles. Kinesin was detected in purified organelle fractions, including synaptic vesicles, mitochondria, and coated vesicles, using quantitative immunoblots and immunoelectron microscopy. In contrast, isolated Golgi membranes and nuclear fractions did not contain detectable levels of kinesin. These results demonstrate that the organelle binding capacity of kinesin is selective and specific. The ability to purify membrane-bounded organelles with associated kinesin indicates that at least a portion of the cellular kinesin has a relatively stable association with membrane-bounded organelles in the cell. In addition, immunoelectron microscopy of mitochondria revealed a patch-like pattern in the kinesin distribution, suggesting that the organization of the motor on the organelle membrane may play a role in regulating organelle motility.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.