Comparison of protein and lipid composition of the human platelet α-granule membranes and glycerol lysis membranes

Platelet glycerol lysis membranes and α-granule membranes were compared with respect to protein and lipid composition. Crossed immunoelectrophoresis using antibodies against whole platelets, and sodium dodecyl sulphate polyacrylamide gel electrophoresis, revealed the presence of the glycoproteins IIb and IIIa, myosin and an antigen termed G4 in both membrane fractions. The glycoproteins Ia, Ib and IIIb, in addition to β₂-microglobulin and actin, appeared specific for the glycerol lysis membranes, whereas two antigens, termed G8 and G18, were observed only in the α-granule membranes. The localization of glycoprotein IIa was inconclusive. Comparison with the surface-located proteins revealed that the glycerol lysis membranes represented a reasonable approximation to a plasma membrane preparation. Radioactively labelled immunoprecipitates obtained after crossed immunoelectrophoresis of ¹²⁵I-labelled platelets were cut out and applied to sodium dodecyl sulphate electrophoresis on polyacrylamide slab gels. Autoradiography of the dried gels revealed that antigen G4 represented a protein with an average molecular weight of 146 000 in its unreduced state and 132 000 in its reduced state. Antigen G18 represented a protein of molecular weight 130 000–135 000 in the reduced as well as unreduced state. Quantitation of protein and lipids showed that the α-granule membranes contained about one-third as much cholesterol and 2-times as much protein in relation to phospholipids as compared to the glycerol lysis membranes. No significant difference between the two membrane preparations was found as regards the composition of their phospholipids.     

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrane and of the whole platelet components established in this system.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes.

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuosus polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrance and of the whole platelet components established in this system.     

Isolation and characterization of two individual glycoprotein components from human milk-fat-globule membranes.

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed 'epithelial-membrane glycoprotein' (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.     

Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by shearing or with the detergent Tween-40.

In purification of cell surface antigens an efficient method for preparing membrane from large numbers of cells is needed. Such a method is described for preparing membranes from rat thymocytes after lysis in the non-ionic detergent Tween-40. Cell surface antigens were recovered at a yield of 30-50%, and a purification of 30-40-fold. By contrast enzyme markers for the other cell organelles were present in the membrane fraction in very low yield. The membrane obtained with the detergent method was compared with that resulting from the best of previously describes methods involving cell lysis by shearing. The detergent method compared favourably for simplicity as well as for yield and purification, and both membrane preparations contained similar protein and glycoprotein constituents. The main glycoprotein bands of membranes from thymocytes and thoracic duct lymphocytes were identified after polyacrylamide gel electrophoresis in dodecyl sulphate. In thymocyte membrane, three main bands at apparent molecular weights of 150 000, 84 000 and 25 000 were seen, and of these the 84 000 glycoprotein did not bind to the lentil lectin. In thoracic duct lymphocyte membrane the 25 000 glycoprotein was absent and a band at 95 000 was intensified in comparison with thymocytes.     

Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by shearing or with the detergent Tween-40

In purification of cell surface antigens an efficient method for preparing membrane from large numbers of cells is needed. Such a method is described for preparing membranes from rat thymocytes after lysis in the non-ionic detergent Tween-40. Cell surface antigens were recovered at a yield of 30–50%, and a purification of 30–40-fold. By contrast enzyme markers for the other cell organelles were present in the membrane fraction in very low yield.The membrane obtained with the detergent method was compared with that resulting from the best of previously described methods involving cell lysis by shearing. The detergent method compared favourably for simplicity as well as for yield and purification, and both membrane preparations contained similar protein and glycoprotein constituents.The main glycoprotein bands of membranes from thymocytes and thoracic duct lymphocytes were identified after polyacrylamide gel electrophoresis in dodecyl sulphate. In thymocyte membrane, three main bands at apparent molecular weights of 150 000, 84 000 and 25 000 were seen, and of these the 84 000 glycoprotein did not bind to the lentil lectin. In thoracic duct lymphocyte membrane the 25 000 glycoprotein was absent and a band at 95 000 was intensified in comparison with thymocytes.     

Separation of the proteins of bovine milk-fat-globule membrane by electrofocusing with retention of enzymatic and immunological activity

1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35--7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5'-nucleotidase, 7.15--7.4; alkaline phosphatase, 5.4--5.7; phosphodiesterase, 4.6--4.8; gamma-glutamyl transpeptidase, 4.4--4.55. 3. Fractions after electrofocusing were analyzed by 'fused rocket' immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67 000, 49 500 and 46 000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing.     

A fibronectin-binding glycoprotein from human platelet membranes.

Fibronectin ('cold-insoluble globulin') has been suggested as a possible mediator of platelet adhesion. A fibronectin-binding protein as partially purified from washed solubilized human platelet membranes by affinity chromatography on fibronectin-Sepharose. The isolated protein migrated as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr (relative molecular mass) of approx. 125 000 under reducing conditions. The protein migrated as a dimer in non-reduced gels. The purified protein did not react with immunoglobulins against fibrinogen or fibronectin when tested in crossed immunoelectrophoresis or electroimmunoassay. The protein and purified fibronectin formed a complex that had a significantly faster mobility in crossed immunoelectrophoresis than did native fibronectin. The presence of heparin in the binding-protein-fibronectin mixture resulted in an even faster mobility of the complex, whereas the mobility of native fibronectin was unaffected. Crossed affinoimmunoelectrophoresis of the complex using different lectins suggested that the binding protein is a glycoprotein containing N-acetylglucosamine residues. The complex, but not purified fibronectin, bound to phenyl-Sepharose on crossed hydrophobic-interaction immunoelectrophoresis. The results strongly suggest the presence of a fibronectin-binding glycoprotein in the platelet membrane.     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland.

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential ultracentrifugation. 2. Neurosecretory vesicles were isolated by sucrose-gradient ultracentrifugation and membranes were obtained after hypo-osmotic lysis of the particles. 3. A method is described for the isolation of a preparation of purified neuronal plasma membranes by using a fraction enriched in nerve endings as a starting material. 4. The purity of the subcellular fractions was estimated by enzyme assays and by examination with the electron microscope. 5. On the basis of the results it was estimated that neuronal plasma membranes constitute more than 30% of the protein of the nerve endings and neurosecretory vesicles more than 45% of the total amount of protein in the homogenate. 6. The proteins of membranes of neurosecretory vesicles and of plasma membranes were solubilized by means of sodium dodecyl sulphate. Polyacrylamide-gel electrophoresis of such preparations showed that both membranes contained a large number of proteins, including three glycoproteins.     

Solubilization and fractionation of glycoproteins and glycolipids of KB cell membranes

1. A fraction enriched in plasma membranes of human tumour KB cell line, a permissive cell for adenovirus type 5, was obtained. 2. Electrophoresis of the membranes in polyacrylamide gels with buffers containing sodium dodecyl sulphate showed that the membranes after reduction with 2-mercaptoethanol contained over 20 polypeptide species. Three polypeptides were glycosylated and had apparent mol.wts. of 92000, 72000 and 62000. 3. The glycoproteins and the specific receptors responsible for adenovirus adsorption to the membranes were readily extracted into solutions containing low concentrations of Triton X-100. Glycolipids and proteins were also made soluble. A membranous residue obtained after Triton X-100 extraction was enriched in several proteins that appeared to consist of polypeptides of lower molecular weight than the average of KB membrane polypeptides. 4. Sphingomyelin, cholesterol and triglycerides were similarly concentrated in the insoluble residue remaining after successive extractions of KB membranes with Triton X-100. Further, ceramide trihexoside was significantly less easily extracted from KB membranes than lactosyl ceramide. 5. The differences noted in the ease of extraction of membrane components are discussed. 6. The components of membranes made soluble by detergent extraction and containing the large part of the KB membrane glycoproteins were subjected to chromatography on Sepharose 6B and DEAE-cellulose and to isoelectric focusing in the presence of buffers containing Triton X-100. In general, the degree of separation into fractions enriched in individual glycoproteins was disappointing. Possible reasons for the poor fractionation of membrane components by chromatographic systems conveniently used for purification of proteins and glycoproteins of non-membranous origin are briefly discussed.     

Comparative study of the major glycoproteins of the plasma membrane and secretory granule membranes of porcine platelets

The major glycoproteins of porcine platelet plasma membranes and alpha-granule membranes were compared. Significant cross-contamination of the two preparations was ruled out by surface labeling and proteolysis experiments. At least four of the major glycoproteins of the alpha-granule membrane had counterparts in the plasma membranes with identical molecular weight and lectin-binding properties. Two of these (mol. wts 110,000 and 125,000) were further analysed by one-dimensional peptide mapping. The results confirmed that there are two distinct pools of identical glycoproteins: one on the surface membrane and the other on the alpha-granular membrane. The 110,000 and 125,000 mol. wt glycoproteins are probably equivalent to glycoproteins IIb and IIIa of the human platelet and may therefore be involved in fibrinogen binding.     

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.     

Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.     

Organisation of the proteins of the chromaffin granule membrane

The organisation of the protein components of bovine chromaffin granules has been investigated by labelling or digesting intact granules or broken membranes with the following reagents: lactoperoxidase/Na¹²⁵I as a reagent for tyrosine residues, $\text{N-}\text{(iodoacetylaminoethyl)-5-naphthylamine-1-sulphonic}$ acid as a reagent for cysteine residues, pronase, and galactose oxidase/KB³H₄. Following treatment, membranes were purified and washed and proteins were examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Rather more than 60 bands were resolved, of which about 40 were relatively intense and reproducible. The bands were classified according to their molecular weights and sensitivity to reagents. Penetration of the membranes by the reagents was assessed by examination of intragranular proteins.The majority of chromaffin granule membrane polypeptides became labelled when intact granules were treated with impermeant reagents. Eleven were probably protected in the intact granules, reactive sites becoming exposed only on membrane lysis. By contrast, carbohydrate moieties of glycoproteins appear to be exposed only on the matrix side of the membrane. Two proteins were shown to span the membrane, although this is probably an underestimate.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.