Molecular Weight Glycoprotein Research Articles

Transferrin receptor of the rabbit reticulocyte.

A Triton X-100 solubilized macromolecular complex of transferrin and a membrane constituent can be isolated by gel chromatography from rabbit reticulocytes previously incubated with 125I-labeled transferrin. The apparent molecular weight of this complex is close to that of ferritin, or about 445 000. On sodium dodecyl sulfate gel electrophoresis the complex displays two glycoprotein subunits, of molecular weights 176 000 and 95 000 in addition to transferrin. A transferrin-binding fraction with a molecular weight near 400 000, containing these subunits, can also be identified in membranes of nonincubated reticulocytes. The corresponding membrane fraction from mature erythrocytes, which have lost transferrin-binding activity, displays both protein subunits, but the 176 000 molecular weight component fails to give a PAS stain for carbohydrate. Treatment of reticulocytes with Pronase, which destroys the ability of the cells to form specific complexes with transferrin, degrades both components. We believe these results are consistent with the hypothesis that the primary transferrin receptor of the rabbit reticulocyte is a glycoprotein of molecular weight in the range 350 000-400 000, comprised of a combination of two subunits with molecular weights 176 000 and 95 000, respectively. Transferrin-binding activity appears to depend on the carbohydrate moiety of the 176 000 subunit.

Glycoprotein synthesis as a function of epithelial cell arrangement: Biosynthesis and release of glycoproteins by human breast and prostate cells in organ culture

We demonstrate that a technique is available to investigate glycoprotein synthesis in organ cultures of human breast and prostate surgical specimens where the 3-dimensional epithelial cell arrangement remains intact. Malignant breast and prostate epithelium maintained their capacity to synthesize glycoproteins for at least 3 days as followed by the incorporation of [3H]glucosamine into macromolecules. Over 70% of incorporation was by malignant cells as judged by autoradiography. Labeled glycoproteins were released into glandular lumina and consequently into the culture fluid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed predominantly one group of macrmolecules released with an apparent molecular weight of 48,000 +/- 6,000 daltons. This glycoprotein was found in all of the breast specimens studied, which included 1 medullary, 1 infiltrating lobular, and 8 infiltrating duct carcinomas. The pattern was independent of the availability of estrogen receptors. A similar glycoprotein was also observed in the culture media from a Grade I and a Grade II well-differentiated infiltrating prostate carcinoma. Incorporation was below the level of detection in 4 of 6 cases of benign prostatic hyperplasia. A more complex pattern of labeled glycoproteins was found in the media of a Grade II and a Grade III poorly-differentiated prostate carcinoma. The established human mammary carcinoma cell line MCF-7 synthesized and released a similar 48,000 molecular weight glycoprotein but additional components with larger molecular weights were also released. An intriguing interpretation that 3-dimensional tissue integrity restricts some glycoprotein synthesis is discussed. Cells grown in 2-dimensional monolayers could escape from such a topographic restriction and express additional families of glycoproteins.

Isolation, characterization, and radioimmunoassay of murine egasyn, a protein stabilizing glucuronidase membrane binding

Glucuronidase present in lysosomes of mouse liver occurs as the free tetramer, whereas glucuronidase present in endoplasmic reticulum occurs in macromolecular complexes containing one to four molecules of the protein egasyn. Earlier genetic and biochemical studies suggest that these complexes, or M forms, function to stabilize the membrane binding of glucoronidase. The detergent Triton X-100 extracts glucuronidase-egasyn complexes intact and they dissociate in the presence of the detergent deoxycholate or upon heating. We have now purfied egasyn by releasing it from antiglucuronidase immunoprecipitates of M forms under relatively mild conditions, such as treatment with deoxycholate or heating at 50 degrees. Isolated egasyn is a glycoprotein of molecular weight about 64,000 and is not unusually hydrophobic in amino acid composition. Monospecific antibody to egasyn was raised. This antibody showed no cross-reactivity with purified beta-glucuronidase and antibody to glucuronidase failed to react with purified egasyn; however, both antibodies bound to egasyn-glucuronidase complexes. A procedure for the radioimmunoassay of egasyn was developed utilizing egasyn labeled with iodine 125. Most of the antigenic sites of egasyn in homogenates of normal liver are masked after extraction with Triton X-100 and only become immunoreactive after exposure to deoxycholate. After unmasking, mouse liver proved to contain about 56 mug of egasyn/g, nearly all of which is localized to the microsomal fraction. Of this total only about 10% was complexed with glucuronidase, suggesting theat the bulk of the egasyn present may be complexed with other proteins. Mice of the inbred strain YBR, which carry the EgO mutation resulting in the absence of microsomal glucuronidase, lacked immunoreactive egasyn, suggesting that the primary defect in this strain lies in the unavailabililty of agasyn to form complexes. There is now considerable evidence in support of the concept that the microsomal forms of glucuronidase exist in membranes complexed with egasyn and that formation of these complexes is required for maintenance of glucuronidase in membranes. Egasyn may represent one of a class of membrane anchor proteins that each stabilize the membrane binding of a charcteristic set of proteins.

Model for the structure of the gastric mucous gel.

THE surface of the stomach is covered by a layer of mucous gel which protects the underlying mucosa from the harmful, acidic stomach contents. The principal component of the gel has been isolated from pig gastric mucus, purified, and shown to be a glycoprotein of molecular weight 2×106 (Table 1)1,2. This glycoprotein consists of four equal sized subunits (molecular weight 5 × 105) joined by disulphide bridges3,4. Each glycoprotein subunit consists of a protein core, 14% by weight of the glycoprotein, with carbohydrate side chains attached. The protein core consists of two regions, one rich in serine, threonine and proline and bearing all the carbohydrate, the other having an amino acid composition characteristic of a globular protein. The latter contains cystine residues which bridge the four subunits. Mild reducing agents and proteolytic enzymes each split the glycoprotein into four subunits4,5. The carbohydrate chains, approximately 15 residues in a branched structure, account for 82% by weight of the glygoprotein and carry ester sulphate residues. The problem therefore is to explain how such a glycoprotein molecule can associate to form a gel which in vivo is essentially impermeable to proteolytic enzymes and which can act as a barrier to protons probably by supporting a pH gradient.

HEPATITIS-B VACCINE: Safety Criteria and Non-B Infection

Immunogenically active fragments of hepatitis B surface antigen, which are a glycoprotein of molecular weight about 28,000 (gp 28) and a protein of molecular weight about 23,000 (p 23), obtained by known methods by treatment of surface antigen with detergent, are prepared in micelle form substantially free from detergent. The micelles are at least as immunogenic as the unfragmented antigen from which they are derived and are useful in the preparation of hepatitis B vaccine. The detergent is removed by layering the detergent containing fragmentation product on an aqueous buffer containing a sucrose gradient and centrifuging the layered buffer when micelles containing both gp 28 and p 23 form and can be recovered in substantially detergent free form from the centrifuged layered buffer.

Hypersensitivity to tobacco antigen.

A glycoprotein of molecular weight 18,000 was purified from saline extracts of cured tobacco leaves by ammonium sulfate fractionation, chromatography on Sephadex G-25 and continuous-flow preparative electrophoresis on polyacrylamide gel. Twelve of 31 volunteers (6/15 smokers and 6/16 nonsmokers) exhibited immediate cutaneous hypersensitivity (wheal and flare reactions) when injected intracutaneously with 2 mug of this material. Immunochemically similar material was demonstrated in, and purified from, cigarette smoke condensate and cigarette smoke. The concentration in cigarette smoke condensate ("tar") was determined to be 1.8-3.6 mg/g. Antigenically crossreactive material was also demonstrated in eggplants, green peppers, potatoes, and tomatoes, which, like tobacco, are members of the family Solanaceae.

Polypeptides of the mouse mammary tumor virus: II. Identification of two major glycoproteins with the viral structure

In order to localize the major structural polypeptides of the murine mammary tumor virus (MuMTV), three subviral fractions were prepared, examined by electron microscopy, and analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. Treatment of intact virus with 0.05 N HCl at 4° for 20 min resulted in the removal of the characteristic surface projections of the virus. Viral membrane fragments with attached projections (rosettes), consisting of stalks and knobs, were prepared by treating MuMTV with Tween-80 and ether. The buoyant density (in sucrose gradients) of the particles after removal of the projections was 1.181 g/cm 3, as compared with 1.171 g/cm 3 for the intact virus. PAGE analysis of the viral material released by the action of acid (for 20 min at 4°) revealed that it contained mainly two glycoproteins of 55,000 (gp55) and 68,000 (gp68) molecular weight (MW), gp55 being the major species. The polypeptide composition of the projectionless particles was found to be complex; the major component was a glycoprotein of 34,000 molecular weight (gp34). Although intact virus contained only one glycoprotein (gp55) in the 50,000–60,000-MW range, the projectionless particles were found to contain two minor components within the same MW range, only one of which was a glycoprotein. The nonglycopeptide component with an apparent MW of 51,000 (p51) appears to be a normal minor protein constituent of MuMTV. The viral rosettes were found to contain gp34, gp55, gp68, and an additional glycoprotein of MW 90,000 (gp90). A comparison of the morphology and polypeptide composition of the intact virus and subviral components suggests that the knobs of the viral projections are composed of gp55. The gp34 component is associated with the viral membrane and may be situated within the membrane bilayer from which the projections protrude.

Taxis to a conjugation-inducing substance in the ciliate Blepharisma.

FOR conjugation of Blepharisma intermedium to take place interaction of soluble gamones with the cells is essential for union between complementary mating types I and II (refs 1 and 2). Type I cells excrete gamone 1 which transforms type II cells so that they can unite. Type II cells excrete gamone 2 which similarly transforms type I cells. Moreover, each gamone promotes the production of the other. Gamone 1, blepharmone, is a glycoprotein of molecular weight 20,000 (refs 3 and 4). Gamone 2, blepharismone, is calcium 3-(2′-formylamino-5′-hydroxybenzoyl) lactate5.

Basic and Clinical Aspects of the Alpha1-Antitrypsin

A deficiency of the major serum alpha1-globulin, the alpha1-antitrypsin, was first described in five patients by Laurell and Eriksson in Sweden in 1963. It soon became obvious that severe alpha1-antitrypsin deficiency was familial, and highly associated with chronic lung disease, having its onset in the third or fourth decade of life. Since the early descriptions of this common deficiency state, it has become clearly associated with familial emphysema in some families, familial infantile cirrhosis in others, and occasionally with a combination of childhood lung and liver disease in siblings. For the pediatrician, severe alpha1-antitrypsin deficiency now enters into the differential diagnosis of both chronic pulmonary disease in childhood and obstructive jaundice in the newborn period; In addition, low levels of alpha1-antitrypsin in serum are characteristic of respiratory distress syndrome, and elevations of this protein may be found in a variety of clinical situations. The, alpha1-antitrypsin probably functions as a major control protein against the tissue-damaging effects of both endogenous and exogenous enzymes. This review will cover several basic and clinical features of this protein with respect to its importance in pediatrics.

Dimeric and Monomeric Forms of HL-A Antigens Solubilized by Detergent

Abstract HL-A antigens solubilized by papain contain two non-covalently linked subunits, a 34,000 m.w. glycoprotein and an 11,000 m.w. protein (1–3), estimated by sodium dodecyl sulfate (SDS)2 gel electrophoresis. The glycoprotein exhibits differences in electrophoretic mobility depending on HL-A specificity (3) and probably bears the antigenic determinant (4). The small protein, when assessed by immunologic and electrophoretic techniques, is identical to β2-microglobulin (4–7), a protein found in serum and in elevated amounts in nephrotic urine, which is significantly homologous to the constant region domains of IgG (8, 9). Numerous studies with immunofluorescent techniques have recently shown that β2-microglobulin is also associated with HL-A antigens on the lymphocyte membrane (10–13), and partially purified HL-A antigens solubilized by detergents contain β2-microglobulin (7) together with a glycoprotein of molecular weight 43,000 to 44,000 (14). In this report, we present evidence that the detergent-solubilized HL-A-β2-microglobulin complex, both labeled with radioactive precursors and in large-scale unlabeled preparations, exists in part in a dimeric form.

Cross-linking of the spike glycoproteins in Semliki Forest virus with dimethylsuberimidate

The bifunctional reagent dimethylsuberimidate was used to cross-link the proteins in: (1) intact Semliki Forest virus (SFV); (2) SFV containing a small amount of the detergent Triton X-100; (3) detergent released SFV membranes; (4) membranes solubilized with Triton X-100 into lipoprotein-detergent complexes; and (5) membranes solubilized into lipid-free glycoprotein-detergent complexes. Analyses of the cross-linked glycoprotein polymers by SDS gel electrophoresis showed that the two 50,000 molecular weight (MW) glycoproteins E1 and E2 of the SFV membrane were preferentially cross-linked into dimers in all preparations. This suggests that oligomeric glycoprotein units, containing two 50,000 MW glycoproteins, are present in the viral membrane and that these remain associated with each other when solubilized with Triton X-100.

Papain-solubilized HL-A Antigens: CHROMATOGRAPHIC AND ELECTROPHORETIC STUDIES OF THE TWO SUBUNITS FROM DIFFERENT SPECIFICITIES

Papain-solubilized HL-A antigens contain two subunits, a glycoprotein of molecular weight 30,000 to 31,000 and a small protein of molecular weight 11,000 to 12,000. These subunits have been studied in six different specificities (HL-A2, HL-A3, HL-A7, HL-A10, HL-A12, and W-27) obtained from normal peripheral lymphocytes and from cultured lymphoblastoid lines. The electrophoretic and chromatographic mobility of the small subunits in a variety of systems was identical, but the large glycoprotein subunits of several specificities could be distinguished by electrophoresis in urea gels at either pH 8.7 or at pH 4.5. It is particularly noteworthy that HL-A7 and W-27 which show a high degree of immunological cross-reactivity were nevertheless distinguishable by electrophoresis at pH 8.7.

Virus-Like Particles in an Extranuclear Mutant of Neurospora crassa

A particulate fraction not present in wild-type cells has been isolated from the cytoplasm and from lysed mitochondria of the respiratory-deficient extranuclear mutant "abnormal-1" of Neurospora crassa. The particles have a density between 1.13 and 1.2 g/ml. They appear in thin sections after OsO(4) fixation as virus-like polymorphic vesicles containing an electron-dense nucleoid of 120-170 nm in diameter. The central core is surrounded by one or two "unit membrane" envelopes of 100 A thickness. The particles contain a single-stranded 33S RNA that is converted to 7-9S RNA by heat treatment in the presence of sodium dodecyl sulfate and that differs in its base composition from mitochondrial and cytoplasmic ribosomal RNA. They contain 7% phospholipids rich in phosphatidylethanolamine and two major proteins, a lipoprotein of molecular weight 15,000 and a glycoprotein of molecular weight 95,000. It is suggested that these virus-like particles originate within mitochondria.

Studies on the Characterization of the Sodium-Potassium Transport Adenosine Triphosphatase: X. PURIFICATION OF THE ENZYME FROM THE RECTAL GLAND OF SQUALUS ACANTHIAS

Abstract Membranes have been isolated from fresh rectal glands of the dogfish shark, Squalus acanthias, in which the specific activity of the (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) is as high as 400 µmoles of Pi per mg of protein per hour. Membranes isolated from frozen rectal glands have only one-fourth to one-third this specific activity. Solubilization of membranes from frozen glands with the nonionic detergent Lubrol WX activates the NaK ATPase 2-fold, but there is no activation of the enzyme with membranes from fresh glands. Purification of Lubrol extracts by zonal centrifugation and a novel ammonium sulfate fractionation gives an enzyme preparation with a specific activity of 1,500 µmoles of Pi per mg of protein per hour. The same specific activity and gel pattern is obtained with enzyme purified from membranes from either fresh or frozen glands. The loss of Lubrol activation of the enzyme from frozen glands occurs on zonal centrifugation, where free Lubrol is removed. The over-all yield of enzyme from the membrane stage is 70%. A mince of 10 rectal glands weighing about 10 to 15 g fresh weight yields about 20 to 30 mg of purified enzyme. The enzyme is completely stable at 0° for 10 days in either the membranous form or in the most highly purified form. It is stable on freezing at -70°. Disc gel electrophoresis shows a gradual elimination of proprotein bands on purification. At the final stage of purification, scanning of Coomassie blue-stained gels gives 72% of the protein as the 97,000 molecular weight catalytic subunit, 19% as the 55,000 molecular weight glycoprotein, and 8.5% as the protein running with the tracking dye. The catalytic subunit and the glycoprotein can be isolated in milligram quantities by solubilization and chromatography on Sephadex G-150. About 90% of the protein applied to the column can be recovered. Sephadex chromatography gives a protein composition of 66% for the catalytic subunit, 28% for the glycoprotein, and 5% for the protein running with the tracking dye. The catalytic subunit and the glycoprotein enrich more or less in parallel as purification proceeds. At the last stage of purification, both proteins are found in the form of vesicles, which bear a striking resemblance on negative staining to purified cytochrome oxidase. Electron-translucent rods and rings which are approximately 80 A in diameter are also formed with projections of about 35 to 55 A at regular intervals. These rods and rings resemble reconstituted oligomycin-sensitive mitochondrial ATPase. The projections appear to be more hydrophilic, and this, coupled with their size, suggests that they are the glycoprotein. Incubation of the enzyme with [γ-32P]ATP, magnesium, and sodium gives 4,080 pmoles of acyl phosphate per mg of protein, which is 2 to 3 times higher than the highest levels of phosphorylation previously reported, thus attesting to the high purity of the enzyme preparation (the level of phosphorylation is generally accepted as being proportional to the number of NaK ATPase molecules in the preparation). The turnover number of the preparation is 6,300 min-1, which is within the range found for most NaK ATPase preparations from different organs and species. Arguments are presented for and against the view that the glycoprotein is a subunit of the NaK ATPase. If the catalytic subunit of molecular weight of 97,000 and the glycoprotein of molecular weight of 55,000 are both integral components of the NaK ATPase, the enzyme is 90 to 95% pure. If the 97,000 molecular weight subunit is the only subunit in the enzyme, the present preparation is 66 to 72% pure.

Acoustic NMR in spin-1 systems

The cell surface of G8-1 clonal muscle cells was labelled at different stages of myogenesis by a variety of methods. Lactoperoxidase-catalysed iodination revealed five surface proteins of 205K (daltons), 160K, 70K, 64K, and 53K which were expressed on myoblast cells only. Four components of 150K, 140K, 54K, and 36K were expressed in myotube cultures only. Labelling of cell-surface sialoglycoproteins by periodate-tritiated borohydride detected the same four myotube-specific components of 150K, 140K, 54K, and 36K that were labelled by lactoperoxidase iodination. Cellular glycoprotein expression during myogenesis was detected by staining polyacrylamide gels of separated muscle components with 125I-concanavalin A (Con A) or 125I-wheat germ agglutinin (WGA). 125I-Con A identified seven glycoproteins of molecular weights, 89K, 76K, 74K, 61K, 52K, 35K, and 27K, which were synthesised in myoblasts and in myotubes at an early stage of differentiation and whose synthesis was then switched off. 125I-WGA detected a myoblast-specific glycoprotein of 100K and a glycoprotein of 89K which was not synthesised in mature myotubes. Fibronectin was labelled by each of the four probes and a similar pattern of regulation was found with each. Fibronectin is synthesised in low amounts in myoblasts. Its level increases with cell fusion and myotube formation to a maximum at the first myotube time point. Its level then falls as the myotubes differentiate. A similar mode of regulation was found with the major Con A receptor (48K) and the major WGA receptor (82–87K).

Dimethylbenzanthracene binding to epidermal chalone.

THE substance called epidermal chalone contained in normal mammalian epidermis is known to inhibit mitosis in a tissue specific manner1–3. The chalone is heat labile and nondialysable; it is precipitated by ethanol and can be preserved in a lyophilized form4. It appears5 to be a glycoprotein of molecular weight between 25,000 and 30,000. Aqueous extracts and a partially purified ethanol fraction of the skin of man, mouse, cod and pig inhibit mitosis in vitro and in vivo4,6,7. Similar extracts from other tissues do not affect mitosis in the epidermis8–12.

Some properties of a lipase from Rhizopus arrhizus separation of a glycopeptide bound to the enzyme

The exocellular lipase from Rhizopus arrhizus is a glycoprotein of molecular weight around 43 000, containing 13–14 molecules of mannose and 2 molecules of hexosamine and a single N-terminal aspartic acid residue (or asparagine). This lipase (lipase I) consists of 2 apparently noncovalently linked portions: a glycopeptide of molecular weight around 8500 and the enzyme protein. This latter can be easily freed from the glycoprotein by heating or by precipitation in the cold with 5% trichloroacetic acid. The glycopeptide does not play any role in the catalytic function of the enzyme. Upon long storage of lipase I, this glycopeptide separates and lipase I is converted into a second active form, lipase II. This slow separation is paralleled by a fragmentation of the glycopeptide into peptides and shorter glycopeptides. The enzymic protein also seems to be partly degraded as shown by the disappearance of the single N-terminal aspartic acid (or asparagine) residue and the appearance of several other N-terminal residues in non-stoichiometric proportions.

Isolation of gastroferrin from human gastric juice

A method is reported for the isolation of gastroferrin - the high molecular weight iron-binding fraction of human gastric juice. This method is based on the ability of the iron-gastroferrin complex to be removed from solution by coprecipitation with ferric hydroxide. When iron is removed from the iron-gastroferrin complex, the gastroferrin is shown to regain its iron chelating ability. Preliminary examination of the product so obtained showed it to be a glycoprotein of molecular weight 260,000. It consists of approximately 85% carbohydrate and 15% protein. Glucosamine, galactosamine, galactose, fucose, and sialic acid were the only carbohydrate residues detected. Serine, threonine, and proline predominated among the 17 amino acids observed; aromatic and sulfur containing amino acids were present in only small amounts.