scholarly journals Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A.

1986 ◽  
Vol 102 (5) ◽  
pp. 1797-1812 ◽  
Author(s):  
R A Bloodgood ◽  
M P Woodward ◽  
N L Salomonsky
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
High Molecular Weight ◽  
Concanavalin A ◽  
Live Cells ◽  
Surface Phenomenon ◽  
Carbohydrate Binding ◽  
Membrane Glycoproteins ◽  
Western Blots ◽  
Flagellar Membrane

Two carbohydrate-binding probes, the lectin concanavalin A and an anti-carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross-react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.

scholarly journals The transmembrane signaling pathway involved in directed movements of Chlamydomonas flagellar membrane glycoproteins involves the dephosphorylation of a 60-kD phosphoprotein that binds to the major flagellar membrane glycoprotein.

1994 ◽  
Vol 127 (3) ◽  
pp. 803-811 ◽  
Author(s):  
R A Bloodgood ◽  
N L Salomonsky
Keyword(s):  
Monoclonal Antibody ◽  
Signaling Pathway ◽  
Live Cells ◽  
Cross Linking ◽  
Carbohydrate Binding ◽  
Membrane Glycoprotein ◽  
Channel Blockers ◽  
Membrane Glycoproteins ◽  
Flagellar Membrane

Cross-linking of Chlamydomonas reinhardtii flagellar membrane glycoproteins results in the directed movements of these glycoproteins within the plane of the flagellar membrane. Three carbohydrate-binding reagents (FMG-1 monoclonal antibody, FMG-3 monoclonal antibody, concanvalin A) that induce flagellar membrane glycoprotein crosslinking and redistribution also induce the specific dephosphorylation of a 60-kD (pI 4.8-5.0) flagellar phosphoprotein (pp60) that is phosphorylated in vivo on serine. Ethanol treatment of live cells induces a similar specific dephosphorylation of pp60. Affinity adsorption of flagellar 32P-labeled membrane-matrix extracts with the FMG-1 monoclonal antibody and concanavalin A demonstrates that pp60 binds to the 350-kD class of flagellar membrane glycoproteins recognized by the FMG-1 monoclonal antibody. In vitro, protein phosphatase 2B (calcineurin) removes 60% of the 32P from pp60; this correlates well with previous observations that directed flagellar glycoprotein movements are dependent on micromolar calcium in the medium and are inhibited by calcium channel blockers and calmodulin antagonists. The data reported here are consistent with the dephosphorylation of pp60 being a step in the signaling pathway that couples flagellar membrane glycoprotein cross-linking to the directed movements of flagellar membrane glycoproteins.

scholarly journals Purification to homogeneity of a high molecular weight human B cell growth factor; demonstration of specific binding to activated B cells; and development of a monoclonal antibody to the factor.

1985 ◽  
Vol 162 (4) ◽  
pp. 1319-1335 ◽  
Author(s):  
J L Ambrus ◽  
C H Jurgensen ◽  
E J Brown ◽  
A S Fauci
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
B Cells ◽  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
High Molecular Weight ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
B Cell Growth Factor

High molecular weight B cell growth factor (HMW-BCGF) produced by a T cell line was purified to homogeneity and demonstrated to bind specifically to activated human B cells. A monoclonal antibody to HMW-BCGF was developed that (a) specifically inhibited the activity of HMW-BCGF in enhancing B cell proliferation, (b) specifically bound to HMW-BCGF in Western blots, (c) specifically absorbed HMW-BCGF activity from culture supernatants, and (d) specifically absorbed an internally labeled protein from T-ALL supernatant which comigrates with HMW-BCGF on sodium dodecyl sulfate-polyacrylamide gels. This antibody should help in cloning the gene for HMW-BCGF and further exploring the physiologic roles of HMW-BCGF.

scholarly journals Functional modification of the Chlamydomonas flagellar surface.

1982 ◽  
Vol 93 (1) ◽  
pp. 88-96 ◽  
Author(s):  
R A Bloodgood ◽  
G S May
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Antibody Preparation ◽  
Substrate Interaction ◽  
Rabbit Igg ◽  
Flagellar Membrane ◽  
Flagellar Proteins ◽  
Force Transduction

Chlamydomonas flagella exhibit force transduction in association with their surface. This flagellar surface motility is probably used both for whole cell gliding movements (flagella-substrate interaction) and for reorientation of flagella during mating (flagella-flagella interaction). The present study seeks to identify flagellar proteins that may function as exposed adhesive sites coupled to a motor responsible for their translocation in the plane of the plasma membrane. The principal components of the flagellar membrane are a pair of glycoproteins (approximately 350,000 mol wt), with similar mobility on SDS polyacrylamide gels. A rabbit IgG preparation has been obtained which is specific for these two glycoproteins; this antibody preparation binds to and agglutinates cells by their flagellar surfaces only. Treatment of cells with 0.1 mg/ml pronase results in a loss of motility-coupled flagellar membrane adhesiveness. This effect is totally reversible, but only in the presence of new protein synthesis. The major flagellar protein modified by this pronase treatment is the faster migrating of the two high molecular weight glycoproteins; the other glycoprotein does not appear to be accessible to external proteolytic digestion. Loss and recovery of flagella surface binding sites for the specific antibody parallels the loss and recovery of the motility-coupled flagellar surface adhesiveness, as measured by the binding and translocation of polystyrene microspheres. These observations suggest, but do not prove, that the faster migrating of the major high molecular weight flagellar membrane glycoproteins may be the component which provides sites for substrate interaction and couples these sites to the cytoskeletal components responsible for force transduction.

scholarly journals Platelet protein organization: analysis by treatment with membrane- permeable cross-linking reagents

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 502-513 ◽  
Author(s):  
GE Davies ◽  
J Palek
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cytoskeletal Proteins ◽  
Cross Linking ◽  
Membrane Glycoproteins ◽  
Platelet Protein ◽  
High Molecular Weight Material ◽  
Organization Analysis ◽  
Thrombin Activation

Abstract We have examined platelet protein organization by treatment of intact resting or thrombin-activated platelets with two cross-linking reagents, diamide or dithiobis(succinimidyl propionate) (DTSP). Cross- linked complexes were separated by polyacrylamide gel electrophoresis in the absence of reducing agent and their composition determined after reductive cleavage and analysis in a second-dimensional gel. The most prominent cross-linked species produced by diamide treatment of of resting platelets are (A) cytoskeletal protein homopolymers, such as myosin heavy chain dimer and actin oligomers, and (B) high molecular weight material consisting of homo- or heteropolymers of cytoskeletal proteins and 230,000, 170,000, 100,000, 55,000, and 52,000 dalton proteins. DTSP treatment forms similar complexes and also cross-links membrane glycoproteins IIb and III into high molecular weight material. Thrombin activation of platelets before treatment with diamide or DTSP results in increased cross-linking of myosin and increased incorporation of several proteins, particularly myosin and glycoproteins IIb and III, into high molecular weight material. The results provide evidence for reorganization of cytoskeletal and membrane proteins during platelet function.

Sign in / Sign up

Export Citation Format

Share Document