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 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 PADGEM protein in human erythroleukemia cells

Blood ◽  
1989 ◽  
Vol 73 (3) ◽  
pp. 722-728 ◽  
Author(s):  
E Yeo ◽  
BC Furie ◽  
B Furie
Keyword(s):  
Molecular Weight ◽  
Binding Sites ◽  
Protein A ◽  
Flow Cytometric Analysis ◽  
Surface Expression ◽  
Membrane Glycoproteins ◽  
Surface Binding ◽  
Cell Content ◽  
Granule Membrane ◽  
Hel Cells

Abstract PADGEM protein, a platelet alpha granule membrane glycoprotein with a molecular weight of 140,000, is translocated to the plasma membrane during granule secretion and platelet activation. PADGEM protein is expressed on the surface of activated platelets but not on the surface of resting platelets. Human erythroleukemia (HEL) cells contain platelet alpha granule-like organelles, alpha granule proteins, and express platelet membrane glycoproteins GPIIb/IIIa and GPIb. We demonstrate that HEL cells express a protein that has a molecular weight identical to that of PADGEM and binds to anti-PADGEM antibodies. The exposure of HEL cells in culture to dimethylsulfoxide (DMSO) increased the number of cells expressing PADGEM. Fluorescence activated flow cytometric analysis demonstrated an increase in mean surface expression of PADGEM in DMSO-exposed cells compared to noninduced cells. Total cell content of PADGEM was increased 5.3-fold after DMSO exposure, as determined by radioimmunoassay. Direct binding experiments with the monoclonal anti-PADGEM antibody KC4 demonstrated specific, saturable, and time-dependent interaction of KC4 with HEL cells. A Kd of 7 nM was estimated. There were 14,000 surface binding sites per cell in noninduced cells and 24,000 surface binding sites per cell in DMSO- induced HEL cells. Surface expression of PADGEM protein on HEL cells was not increased with platelet agonists, including thrombin, epinephrine, ADP, nor cytokines, including IL-1, IL-2, tissue necrosis factor. The presence of PADGEM protein in HEL cells should facilitate the elucidation of the function of PADGEM protein.

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.

High Molecular Weight Kininogen Is Cleaved by FXIa at Three Sites: Arg409-Arg410, Lys502-Thr503 and Lys325-Lys326

2000 ◽  
Vol 83 (05) ◽  
pp. 709-714 ◽  
Author(s):  
T. Mauron ◽  
B. Lämmle ◽  
W. A. Wuillemin
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Coagulation Factor ◽  
Cleavage Sites ◽  
High Molecular Weight Kininogen ◽  
Surface Binding ◽  
Amino Acid Sequence Analysis ◽  
Sds Page ◽  
System Activation

SummaryWe investigated the cleavage of high molecular weight kininogen (HK) by activated coagulation factor XI (FXIa) in vitro. Incubation of HK with FXIa resulted in the generation of cleavage products which were subjected to SDS-Page and analyzed by silverstaining, ligandblotting and immunoblotting, respectively. Upon incubation with FXIa, bands were generated at 111, 100, 88 kDa on nonreduced and at 76, 62 and 51 kDa on reduced gels. Amino acid sequence analysis of the reaction mixtures revealed three cleavage sites at Arg409-Arg410, at Lys502-Thr503 and at Lys325-Lys326. Analysis of HK-samples incubated with FXIa for 3 min, 10 min and 120 min indicated HK to be cleaved first at Arg409-Arg410, followed by cleavage at Lys502-Thr503 and then at Lys325-Lys326.In conclusion, HK is cleaved by FXIa at three sites. Cleavage of HK by FXIa results in the loss of the surface binding site of HK, which may constitute a mechanism of inactivation of HK and of control of contact system activation.

Effect of altered oligosaccharide structure on the cell surface number, distribution and turnover of the high molecular weight acidic glycoproteins of CHO cells

1984 ◽  
Vol 67 (1) ◽  
pp. 1-23
Author(s):  
L.A. Fitzgerald ◽  
J.B. Denny ◽  
G.A. Baumbach ◽  
C.M. Ketcham ◽  
R.M. Roberts
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
High Molecular Weight ◽  
Cho Cells ◽  
Carbohydrate Structure ◽  
Membrane Glycoproteins ◽  
Number Distribution ◽  
Mutant Lines ◽  
Mutant Cells ◽  
Surface Glycoproteins

The influence of altered carbohydrate structure on the surface number, distribution and turnover of plasma membrane glycoproteins has been studied in Chinese hamster ovary (CHO) cells by comparing three lines that are resistant to the cytotoxic effects of wheat germ agglutinin (WGA) with parental CHO cells. The glycoproteins investigated were members of a group of high molecular weight acidic glycoproteins (HMWAG). On parental cells these represent the major surface components that become labelled by lactoperoxidase-catalysed iodination. They are the only plasma membrane glycoproteins that bind WGA. The mutant lines also possess iodinatable surface polypeptides of high molecular weight, but these were less acidic and electrophoretically less diffuse than those from parental cells. These polypeptides in general did not bind [125I]WGA when two-dimensional polyacrylamide gels were overlaid with iodinated lectin. Mutant cells treated with fluorescein-conjugated WGA showed low surface fluorescence. However, the nuclear envelope and a small region in the perinuclear zone fluoresced strongly. Together, these results confirm that the surface glycoproteins of mutant cells had altered carbohydrate structure. Mouse antiserum prepared against the HMWAG, however, bound equally effectively to the mutant lines as to the parental lines. Indirect immunofluorescence experiments showed that the HMWAG had a fairly uniform distribution over the surface, and that internalization induced by second antibody occurred at a similar rate and in a similar manner in all lines, including the mutants. Electron microscopic observations using immunoperoxidase procedures confirmed the similarities in glycoprotein distribution on mutant and parental cells. Two mouse monoclonal antibodies raised against the HMWAG also revealed no difference in the number or topography of surface glycoproteins. Finally, the half-lives of several HMWAG in a parental and a mutant line (15B) maintained on low-serum medium were compared by means of a 125I/131I double-label technique. Half-lives of HMWAG from the former averaged 12 h and from the latter 11 h. It is concluded that the lack of complex termini on oligosaccharides of this particular group of CHO plasma membrane glycoproteins has no effect on their number, distribution or turnover.

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

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.

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