scholarly journals Identification by monoclonal antibodies and characterization of human platelet caldesmon.

1986 ◽  
Vol 102 (5) ◽  
pp. 1748-1757 ◽  
Author(s):  
J Dingus ◽  
S Hwo ◽  
J Bryan
Keyword(s):  
Molecular Weight ◽  
Smooth Muscle ◽  
Monoclonal Antibodies ◽  
Cultured Cells ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Binding Activity ◽  
Actin Binding ◽  
Dependent Manner ◽  
Platelet Protein

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.

Phosphorylation Of Platelet Actin-Binding Protein During Platelet Activation

1981 ◽  
Author(s):  
Roger C Carroll ◽  
Jonathan M Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Platelet Protein ◽  
External Calcium

We have followed the 32P-labelling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples we found 2 to 3 fold labelling increases in actin-binding protein 30 to 60 seconds after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis of purified actin-binding protein as well as cytoskeletal cores prepared by detergent extraction of activated 32p-iabelled platelets. In addition, these cytoskeletal cores indicated that the 32P-labelled actin-binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labelling of actin-binding protein over an 8 minute time course revealed that in aggregating platelet samples rapid desphosphorylation to almost initial levels occurred between 3 and 5 minutes. A similar curve was obtained for the 20,000 apparent molecular weight peptide. This rapid dephosphorylation was shown to be dependent on platelet aggregation in the absence of external calcium or in thrombastenic platelets lacking the aggregation response to activation. These results suggest that phosphorylation of actin-binding protein initiates its association with the platelet cytoskeleton during activation.

scholarly journals Isolation and function of a human endothelial cell C1q receptor

1993 ◽  
Vol 2 (6) ◽  
pp. 447-452 ◽  
Author(s):  
M. R. Daha ◽  
L. Dunn ◽  
R. van den Berg ◽  
Y. Muizert-de Lange ◽  
A. Gerritsen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Primary Immune Response ◽  
Reduced Form ◽  
Human Umbilical Cord ◽  
Dependent Manner ◽  
Western Blots

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 × 105binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1–5 × 109human umbilical cord EC by affinity chromatography on C1q–Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q–Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC–TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55–62 kDa in the unreduced state and a molecular weight of 64–68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of125I-C1q to endothelial cells but F(ab')2anti-C1qR mAb inhibited the binding of a125I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54–60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC–C1qR.

scholarly journals Platelets contain proteins immunologically related to red cell spectrin and protein 4.1

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 52-59
Author(s):  
GE Davies ◽  
CM Cohen
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Cell Protein ◽  
Immunoperoxidase Staining ◽  
Red Cell ◽  
Protein 4.1 ◽  
Platelet Protein

Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.

scholarly journals Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 466-471 ◽  
Author(s):  
RC Carroll ◽  
JM Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Cell Contact ◽  
Actin Binding Protein ◽  
Platelet Protein

Abstract In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

scholarly journals Platelets contain proteins immunologically related to red cell spectrin and protein 4.1

Blood ◽  
1985 ◽  
Vol 65 (1) ◽  
pp. 52-59 ◽  
Author(s):  
GE Davies ◽  
CM Cohen
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Cell Protein ◽  
Immunoperoxidase Staining ◽  
Red Cell ◽  
Protein 4.1 ◽  
Platelet Protein

Abstract Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.

scholarly journals Phosphorylation of platelet actin-binding protein during platelet activation

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 466-471 ◽  
Author(s):  
RC Carroll ◽  
JM Gerrard
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Time Course ◽  
Binding Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Actin Binding ◽  
Cell Contact ◽  
Actin Binding Protein ◽  
Platelet Protein

In this study we have followed the 32P-labeling of actin-binding protein as a function of platelet activation. Utilizing polyacrylamide- sodium dodecyl sulfate gel electrophoresis to resolve total platelet protein samples, we found 2--3-fold labeling increases in actin-binding protein 30--60 sec after thrombin stimulation. Somewhat larger increases were observed for 40,000 and 20,000 apparent molecular weight peptides. The actin-binding protein was identified on the gels by coelectrophoresis with purified actin-binding protein, its presence in cytoskeletal cores prepared by detergent extraction of activated 32P- labeled platelets, and by direct immunoprecipitation with antibodies against guinea pig vas deferens filamin (actin-binding protein). In addition, these cytoskeletal cores indicated that the 32P-labeled actin- binding protein was closely associated with the activated platelet's cytoskeleton. Following the 32P-labeling of actin-binding protein over an 8-min time course revealed that in aggregating platelet samples rapid dephosphorylation to almost initial levels occurred between 3 and 5 min. A similar curve was obtained for the 20,000 apparent molecular weight peptide. However, rapid dephosphorylation was not observed if platelet aggregation was prevented by chelating external calcium or by using thrombasthenic platelets lacking the aggregation response. Thus, cell-cell contact would seem to be crucial in initiating the rapid dephosphorylation response.

scholarly journals Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin.

1985 ◽  
Vol 100 (5) ◽  
pp. 1656-1663 ◽  
Author(s):  
A Bretscher ◽  
W Lynch
Keyword(s):  
Molecular Weight ◽  
Smooth Muscle ◽  
Cultured Cells ◽  
Stress Fibers ◽  
Actin Binding ◽  
Cross Linking ◽  
Light Microscope Level ◽  
Chicken Gizzard ◽  
Heat Stable

Caldesmon is an F-actin cross-linking protein of chicken gizzard smooth muscle whose F-actin binding activity can be regulated in vitro by Ca2+-calmodulin (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, 1981, Proc. Natl. Acad. Sci. USA, 78:5652-5655). It is a rod-shaped, heat-stable, F-actin bundling protein and is the most abundant F-actin cross-linking protein of chicken gizzard smooth muscle presently known (Bretscher, A., 1984, J. Biol. Chem., 259:12873-12880). We report the use of polyclonal antibodies to caldesmon to investigate its distribution and localization in other cells. Using immune blotting procedures, we have detected immunoreactive, heat-stable forms of caldesmon in cultured cells having either approximately the same apparent polypeptide molecular weight as gizzard caldesmon (120,000-140,000) or a substantially lower molecular weight (71,000-77,000). Through use of affinity-purified antibodies in indirect immunofluorescence microscopy, we have localized the immunoreactive forms to the terminal web of the brush border of intestinal epithelial cells and to the stress fibers and ruffling membranes of cultured cells. At the light microscope level caldesmon is distributed in a periodic fashion along stress fibers that is coincident with the distribution of tropomyosin and complementary to the distribution of alpha-actinin.

Binding, degradation, and biological activity of insulin in vascular smooth muscle cells

1985 ◽  
Vol 249 (3) ◽  
pp. E292-E298
Author(s):  
N. Kaiser ◽  
A. Tur-Sinai ◽  
M. Hasin ◽  
E. Cerasi
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Insulin Binding ◽  
Maximal Response ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Igf I

The interaction of insulin with the vascular smooth muscle was studied using cultures derived from the bovine aortic arch. The cultured cells exhibited specific binding of 125I-insulin that was reversible and dependent on pH. Both insulin and insulinlike growth factor (IGF) I competed for 125I-insulin binding; IGF I, however, was less effective than insulin by at least an order of magnitude. Insulin binding was accompanied by internalization and degradation of the hormone in a temperature- and time-dependent manner. Chloroquine and other lysosomotropic agents elevated the internalized insulin and reduced its degradation. Pre-exposure of cell cultures to insulin resulted in downregulation of cell surface receptors. Insulin stimulated alpha-aminoisobutyric acid transport in confluent smooth muscle cells. The maximal response was observed at 100 ng/ml insulin with a half-maximal effect at 10 ng/ml. Sparse, serum-starved smooth muscle cells responded to insulin with a dose-dependent increase in [3H]-thymidine incorporation into DNA. Although the effect was already apparent at 1 ng/ml insulin, it reached near maximal level only at 10,000 ng/ml. IGF I also stimulated DNA synthesis in smooth muscle cells; however, at low concentrations insulin was more efficient in this respect. Human growth hormone was inactive. The data indicate the presence of specific receptors for insulin in bovine aortic smooth muscle cells. These receptors appear to mediate the metabolic activity as well as part of the mitogenic effect of insulin in these cells.

scholarly journals Mapping nucleolar proteins with monoclonal antibodies.

1984 ◽  
Vol 99 (6) ◽  
pp. 1981-1988 ◽  
Author(s):  
J Kistler ◽  
Y Duncombe ◽  
U K Laemmli
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Nucleolar Proteins

Using monoclonal antibodies as probes, we have characterized three antigens with respect to localization in the nucleolus, molecular weight and solubility. Two proteins, of 110,000 and 94,000 apparent molecular weight, were found associated with the ribonucleoprotein fibers. A third protein, with a molecular weight of 40,000, was accumulated at the nucleolar periphery, was present in the nucleoplasm, and may be involved in pre-ribosome maturation and transport.

Characterization Of The ADP-Induced Fibrinogen Btnd1Ng Sites On Human Platelets Using Photoaffinity Techniques: Some Preliminary Findings

1981 ◽  
Author(s):  
Ellinor I Peerschke ◽  
Mariorie B Zucker ◽  
Avner Rotman
Keyword(s):  
Molecular Weight ◽  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Fragment ◽  
Fibrinogen Receptor ◽  
Congenital Deficiency ◽  
Sds Page ◽  
Platelet Membrane Receptor

The interaction of fibrinogen with its, platelet membrane receptor was investigated using 125-labeled fibrinogen which was photoaffinity labeled with a light-sensitive azide. This photoreactive material (125I-NPA-fibr) was indistinguishable from unlabeled fibrinogen as well as from iodinated fibrinogen on SDS-PAGE. It bound specifically to platelets stimulated with ADP and was crosslinked to the platelet membrane after exposure to light ( λ >300 nm) for 4 min. No crosslinking was observed in the presence of EDTA or with platelets that failed to aggregate with ADP either due to the congenital deficiency thrombasthenia or following incubation with EDTA for 8 min at 37° , pH 7.8 and recalcification. SDS-PAGE of platelets bearing crosslinked 125I-NPA-fibr revealed a radiolabeled band of about 450,000 daltons in addition to the 340,000 dalton radioactive band of fibrinogen, suggesting that fibrinogen had been covalently bound to a platelet membrane component with an intact apparent molecular weight of approximately 110,000 daltons. Following reduction, an extra radioactive band was noted at 80,000 daltons. As the A∝-chains of fibrinogen were too weakly labeled to be detected by autoradiography, this indicated that either the Bβ or γchain of fibrinogen was attached to a 25,000-35,000 molecular weight platelet membrane fragment. We conclude that the additional radioactive bands observed after electrophoresis of platelets bearing specifically bound-photoaffinity labeled 125I-fibrinogen most likely represent the binding of the B β or γ chains of fibrinogen to the platelet fibrinogen receptor which may be GPIIb.

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