scholarly journals Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells.

1986 ◽  
Vol 261 (14) ◽  
pp. 6137-6140
Author(s):  
A R Safa ◽  
C J Glover ◽  
M B Meyers ◽  
J L Biedler ◽  
R L Felsted
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Surface Membrane ◽  
Photoaffinity Labeling ◽  
Multidrug Resistant ◽  
Membrane Glycoprotein ◽  
Resistant Cells

CA2+-INDUCED STRUCTURAL TRANSITIONS OF THE PLATELET GP IIb-IIIa COMPLEX

1987 ◽  
Author(s):  
B Steiner ◽  
D R Phillips
Keyword(s):  
Molecular Weight ◽  
Platelet Aggregation ◽  
High Molecular Weight ◽  
Functional Activity ◽  
Sucrose Gradient ◽  
Membrane Glycoprotein ◽  
Structural Transitions ◽  
Heterodimeric Complex ◽  
Triton X ◽  
Active Subunits

Previous studies have shown that the membrane glycoprotein (GP) IIb-IIIa complex can be reversibly dissociated by incubating platelets for 5 min at 37°C in an EDTA-containing buffer. Prolonged incubations (30 min) with EDTA, however, result in the formation of high molecular weight aggregates of GP IIb and GP IIIa. These aggregates of individual GP's neither bind fibrinogen nor support platelet aggregation, indicating that chelation of Ca2+ can affect the functional activity of GP IIb-IIIa. The present study was designed to identify conditions for the generation of functionally active GP IIb and GP IIIa. Functionally active subunits were defined as those which reformed GP IIb-IIIa complexes. The complexes were quantified by sucrose gradient sedimentation (complexed, dissociated and aggregated GP’s have different sedimentation coefficients) and thrombin hydrolysis (dissociated and aggregated GP lib are susceptible to hydrolysis by thrombin while GP lib in the GP IIb-IIIa complex is thrombin resistant). Purified GP IIb-IIIa could be dissociated by a 5 min incubation at 37°C with ≤ 10−5 M Ca2+. When the complexes were dissociated in the presence of Ca2+ concentrations below 10−6 m, the monomeric GP IIIa was converted to a slower sedimenting form; this change in structure caused it to become functionally inactive. In the presence of very low Ca2+ concentrations 10−6 M) both dissociated subunits subsequently formed high molecular weight aggregates. However, these changes in structure and loss in function could be prevented by dissociating the complexes in 10−6 M Ca2+ and immediately readding raM Ca2+ at 4°C. When this solution was warmed to 20°C, almost 70% of the dissociated subunits reformed heterodimeric complexes. Storage at 4°C for as long as 6 h did not alter the functional activity of these subunits. Octylglucoside, but not Triton X-100, completely inhibited reassociation. Experiments performed in the presence of various H+ and salt concentrations showed that the interactive forces between GP IIb and GP IIIa are both electrostatic and hydrophobic. Thus, conditions have been obtained for the preparation of functionally active GP IIb and GP IIIa which can reform the native heterodimeric complex. Various Ca2+ concentrations can have multiple effects on the structure of the dissociated subunits.

scholarly journals Fibronectin-independent adhesion of fibroblasts to the extracellular matrix: mediation by a high molecular weight membrane glycoprotein.

1981 ◽  
Vol 91 (3) ◽  
pp. 647-653 ◽  
Author(s):  
P A Harper ◽  
R L Juliano
Keyword(s):  
Molecular Weight ◽  
Extracellular Matrix ◽  
High Molecular Weight ◽  
Cho Cells ◽  
Matrix Material ◽  
Differential Sensitivity ◽  
Galactose Oxidase ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Type Ii

Fibroblastic CHO cells readily adhere to fibronectin (Fn) coated substrata. From the parental cell population we have recently selected a series of adhesion variants (ADV cells) that cannot adhere to Fn substrata (Harper and Juliano. 1980. J. Cell. Biol. 87:755-763). However, ADV cells readily adhere to substrata coated with extracellular matrix material (ECM) derived from human diploid fibroblasts by a mechanism that does not involve fibronectin (Harper and Juliano. 1981. Nature (Lond.). 290:136-138). Te Fn-dependent adhesion mechanism of parental cells (type 1 adhesion) and the ECM-dependent adhesion of ADV cells (type II adhesion) can also be discriminated on the basis of their differential sensitivity to proteolysis, with the type II mechanism being far more sensitive. In this communication we report that parental CHO cells possess both type I and type II mechanisms whereas ADV cells possess only the type II mechanism. We also identify a high molecular weight membrane glycoprotein (gp 265) that seems to play a role in type II adhesion. This component is detected by [125I]lactoperoxidase of [3H]borohydride-galactose oxidase labeling of surface proteins in WT and AD cells. Cleavage of gp 265 with low doses of proteases correlates completely with the loss of type II adhesion capacity. Thus CHO cells possess two functionally and biochemically distinct adhesion mechanisms, one involving exogenous Fn and the other mediated by the membrane component gp 265.

scholarly journals Surface membrane glycoproteins of wild-type and differentiation-inducer- resistant HL-60 cells

Blood ◽  
1985 ◽  
Vol 66 (3) ◽  
pp. 606-613
Author(s):  
RL Felsted ◽  
SK Gupta ◽  
CJ Glover ◽  
RE Gallagher
Keyword(s):  
Molecular Weight ◽  
Surface Membrane ◽  
Resistance Mechanism ◽  
Cytotoxic Agents ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
Purine Bases ◽  
In The Wild ◽  
Differentiation Inducer ◽  
Resistant Cells

Surface membrane glycoproteins (SMGs) of cells from the parental wild- type HL-60 cell line and from three sublines variably cross-resistant to the granulocyte differentiation-inducing effects of retinoic acid (RA), dimethylsulfoxide (DMSO), and certain purine bases (6-thioguanine [6TG] or hypoxanthine) were studied by one-dimensional and two- dimensional gel electrophoresis. After both oligosaccharide (periodate/borotritide) and peptide (1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril) ectolabeling procedures, striking common changes were noted in the gel electrophoretic patterns of the SMGs from the RA- and 6TG-resistant sublines compared to those from the wild-type HL-60 line or the DMSO-resistant subline. Most prominently, this included the presence in the RA- and 6TG-resistant cells of an apparent high molecular weight acidic glycoprotein(s) (mol wt, 200 to 285 kilodaltons [kD]; isoelectric point range [pl], 4.5 to 6.0) not observed in the wild-type or DMSO-resistant cells and, conversely, the presence of a lower molecular weight glycoprotein(s) (mol wt, 120 to 165 kD; pl, 4.2 to 5.9) in the wild-type and DMSO-resistant cells, which was absent or much reduced in the RA- and 6TG-resistant cells. These acidic SMGs did not change as a function of the induction of granulocyte differentiation. However, some other more basic SMGs varied as a function of granulocyte differentiation in both the wild-type and differentiation inducer-resistant sublines, including the loss of the transferrin receptor and the gain of a mol wt 55- to 60-kD neutrophil- associated protein. In the context of previously reported information, these results indicate (1) that the overall pattern of SMG changes in the RA- and 6TG-resistant cells closely resembles that associated with multidrug (pleiotropic) resistance to cytotoxic agents in a variety of mammalian cells; (2) that the RA/6TG resistance-associated SMG changes are not granulocyte differentiation stage-specific; and (3) that either the RA/6TG resistance-associated SMG changes are not related to the resistance mechanism or they are involved in the resistance/cross- resistance mechanism(s) for RA/purine bases but not for DMSO.

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