Human erythrocyte membrane glycoprotein: A re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis

Segrest, Jere P.; Jackson, Richard L.; Andrews, Emily; Marchesi, V.

doi:10.1016/0006-291x(71)90612-7

Cited by 542 publications

(146 citation statements)

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“…Carbohydrates affect the migration of glycoproteins in S D S -P A G E in a complex manner. Chains of uncharged sugar residues decrease the migration of such molecules and can therefore lead to an over-estimation of molecular weight (Segrest et al, 1971), but sialic acid has the opposite effect. It has been found in studies on cell surface glycoproteins that neuraminidase digestion did not affect the mobility of molecules containing fewer than six sialic acid residues per hundred amino acids, whereas similar treatment of a glycoprotein with five times this amount of sialic acid resulted in a change of apparent mol.…”

Section: Discussionmentioning

confidence: 99%

A Structural Investigation of the Epstein--Barr (EB) Virus Membrane Antigen Glycoprotein, gp340

Morgan

Smith

Barker

et al. 1984

Journal of General Virology

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SUMMARYEpstein-Barr (EB) virus membrane antigen (MA) glycoprotein (gp340) purified by a molecular weight-based technique has been subjected to biochemical analysis. Following treatment with glycosidases or tunicamycin during synthesis, the carbohydrate moiety was found to be made up of both O-linked and N-linked types and to constitute about 50~o of the molecular mass. Digestion studies with neuraminidase and oligosaccharidase have indicated that the molecule is heavily sialated with most of the sialic acid located on the O-linked sugars. The high carbohydrate content of gp340 appears to confer resistance to proteolysis, thus, V8 protease was only effective at concentrations above 1 mg/ml when three large fragments of mol. wt. 330K, 190K and 160K were generated. Removal of sialic acid before V8 protease digestion did not alter this pattern nor affect the antigenicity of the digestion fragments. Antigenicity of the intact molecule was likewise unaffected by removal of sialic acid nor were the O-linked and N-linked carbohydrate moieties essential for this property. The binding of virusneutralizing human sera and monoclonal antibody by gp340 from which either Olinked or N-linked sugars had been removed seems to indicate that the sites on the molecule that generate the neutralizing antibodies are present in the protein component. The significance of these results is discussed in relation to the development of a subunit vaccine against EB virus.

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Section: Discussionmentioning

confidence: 99%

A Structural Investigation of the Epstein--Barr (EB) Virus Membrane Antigen Glycoprotein, gp340

Morgan

Smith

Barker

et al. 1984

Journal of General Virology

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“…With respect to the known molecular weights of the erythrocyte ghost proteins (20), the apparent molecular weights of GP-B, GP-A, GP-1, and GP-2 were about 190,000, 130,000, 85,000, and 45,000, respectively. Considering the anomalous electrophoretic behavior of glycoproteins in SDS-polyacrylamide gels (22)(23)(24), these molecular weights should not be taken too seriously.…”

Section: Resultsmentioning

confidence: 99%

Crosslinking of the Glycoproteins in Human Erythrocyte Membranes

Ji¹

1974

Proc. Natl. Acad. Sci. U.S.A.

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The glycoproteins of human erythrocyte membranes were crosslinked with dimethyl adipimidate dihydrochloride. On sodium dodecyl sulfate-polyacrylamide gel electrophoregrams of the crosslinked solubilized membranes, at least three new glycoprotein complexes appeared in addition to the normal glycoprotein species. One of the new glycoprotein complexes was shown to contain two of the three species of membrane glycopro. teins. Glycoproteins have been shown to be vital in determining the structure and organization of plasma membranes (1-4), and there is evidence suggesting their important role in cell-to-cell contact, adhesion, hormone interaction, and viral transformation (5-12). Little is known concerning the structures of membrane glycoproteins in animal cells, with the exception of glycoproteins from human erythrocyte membranes.Three major species of glycoproteins have been found in human erythrocyte membranes (13); one of these has been implicated in reversible aggregation and dispersion in the membrane plane (2-4). Interactions with other membrane components may be important in determining their lateral movement and, in fact, there are such indications (4). Chemical crosslinking of the glycoproteins to adjacent molecules is one approach to studying their organization and interactions in the membrane. A variety of bifunctional crosslinking reagents were tested for that purpose. None of these reagents was successful in crosslinking glycoproteins, although most or all of the proteins in the membrane have been crosslinked by this technique (14-16). It will be shown here that the glycoproteins and proteins can be crosslinked with dimethyl adipimidate dihydrochloride, and that new glycoprotein complexes can be produced. MATERIALS AND METHODSOut-dated, normal human blood was purchased from the Wyoming Blood Service, Cheyenne, Wyo. Erythrocyte ghosts were prepared as described (17).Dimethyl adipimidate dihydrochloride (DIMA) was prepared according to the method of McElvain and Schroeder (18) and dissolved in a buffer containing 0.9 g/100 ml of NaCl and 5 mM Na2HPO4 (pH 7.5). The erythrocyte ghosts and DMA were mixed in the buffer to make the concentration of ghost proteins 0.15 mg/ml and reagent 0.5 mg/ml. The protein concentration was determined by the modified Lowry method (19). The reaction was stopped by centrifugation and resuspension of the ghosts twice in the buffer.The membrane proteins were solubilized in 1% sodium dodecyl sulfate (SDS), subjected to electrophoresis in SDSpolyacrylamide gels, and stained for either glycoproteins or proteins, as described (20). All of the chemicals necessary to prepare the gels were purchased from the Eastman Kodak Co. The gels were scanned on an ISCO gel scanner at 550 nm.The crosslinking was cleaved by ammonolysis as follows: A zone corresponding to a specific band of crosslinked product in an unstained duplicate gel was cut into about 2-mm pieces and incubated in 100 ,ul of a solution containing 35% ammonium hydroxide, 10% SDS, acetic acid, and 2-mercaptoethanol...

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“…This discrepancy was not unexpected. It is well documented that glycoproteins as well as proteins with rigid disulfide linkages often exhibit anomalous migration profiles in SDS-PAGE (Segrest et al, 1971;Marciani and Papamatheakis, 1978). Additionally, we observed similar discrepancies with M r determination for P. alliacea alliinase, which shares with the P. alliacea LFS four of its five protein subunits (Musah et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Discovery and Characterization of a Novel Lachrymatory Factor Synthase inPetiveria alliaceaand Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds

Musah

Kubec

2009

Plant Physiology

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A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two a-subunits (68.8 kD each), one g-subunit (22.5 kD), and one d-subunit (11.9 kD). The two a-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-L-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.

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Human erythrocyte membrane glycoprotein: A re-evaluation of the molecular weight as determined by SDS polyacrylamide gel electrophoresis

Cited by 542 publications

References 4 publications

A Structural Investigation of the Epstein--Barr (EB) Virus Membrane Antigen Glycoprotein, gp340

A Structural Investigation of the Epstein--Barr (EB) Virus Membrane Antigen Glycoprotein, gp340

Crosslinking of the Glycoproteins in Human Erythrocyte Membranes

Discovery and Characterization of a Novel Lachrymatory Factor Synthase inPetiveria alliaceaand Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds

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