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 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 THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE

1972 ◽  
Vol 55 (2) ◽  
pp. 390-405 ◽  
Author(s):  
Ann L. Hubbard ◽  
Zanvil A. Cohn
Keyword(s):  
Molecular Weight ◽  
Cell Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Chloride Ions ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Double Labeling ◽  
Glucose System

An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane. 97% of the incorporated isotope is in the erythrocyte ghost and 3% is associated with hemoglobin. No significant labeling of the red cell membrane occurs in the absence of LPO or by the deletion of any of the other reagents. A 6 million-fold excess of chloride ions inhibits iodination by no more than 50%. Incorporation of up to 1 x 106 iodide atoms into a single erythrocyte membrane results in no significant cell lysis. The incorporated label is exclusively in tyrosine residues as monoiodotyrosine. 10–15% of the trichloroacetic acid-precipitable radioactivity can be extracted with lipid solvents but is present as either labeled protein or 125I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins reveals only two labeled protein bands out of the 15 present, and the presence of 50-1 x 106 iodide atoms per ghost does not alter this pattern. Component a has a molecular weight of 110,000, is carbohydrate poor, and represents 40% of the total label. Component b has an apparent molecular weight of 74,000, contains all of the demonstrable sialic acid, and accounts for 60% of the total label. Trypsinization of iodinated, intact red cells results in the disappearance of only component b, the appearance of labeled glycopeptides in the medium, and the absence of smaller, labeled peptides remaining in the membrane. Pronase treatment hydrolyzes component b in a similar fashion, but also cleaves component a to a 72,000 mol wt peptide which is retained in the membrane. A combination of protease treatment and double labeling with 125I and 131I does not reveal the appearance of previously unexposed proteins.

Variation in goat fibre protein

1989 ◽  
Vol 40 (3) ◽  
pp. 675 ◽  
Author(s):  
DJ Tucker ◽  
AHF Hudson ◽  
A Laudani ◽  
RC Marshall ◽  
DE Rivett
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Wool Fibre ◽  
Two Dimensional ◽  
Protein Patterns ◽  
Cashmere Goats

The proteins from a range of cashmere, mohair, angoratcashmere crossbred and wool fibre samples were extracted at pH 8 with 8 M urea containing dithiothreitol, and were then radiolabelled by S-carboxymethylation using iodo(2-14C) acetate. The proteins from each sample were examined by two dimensional polyacrylamide gel electrophoresis in which the separation in the first dimension was according to charge at pH 8.9 and in the second dimension according to apparent molecular weight in the presence of sodium dodecyl sulfate. After electrophoresis the proteins were detected by fluorography. Protein differences in keratin samples from some individual goats existed, although the overall protein patterns were similar. None of the differences were consistent with any one goat fibre type. The protein patterns obtained for fibre samples from individual cashmere goats showed some differences when compared to those found for commercial blends from the same country of origin, indicating that blending can mask any animal-to-animal variation. While the electrophoretic technique does not unequivocally distinguish between cashmere, mohair and angora/cashmere crossbred fibres it does differentiate between wool and goat fibres.

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.

Determination of the Molecular Weight and the Hydrodynamic Properties of a Polypeptide from the Thylakoid Membrane by Sedimentation, Diffusion and Binding Measurements in Dodecyl Sulphate Solutions

1975 ◽  
Vol 30 (9-10) ◽  
pp. 615-621 ◽  
Author(s):  
Hans Craubner ◽  
Friederike Koenig ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Partial Specific Volume ◽  
Hydrodynamic Properties ◽  
Lamellar System ◽  
Self Diffusion

The molecular weight and hydrodynamic properties of a polypeptide isolated from the lamellar system of Antirrhinum chloroplasts were determined in sodium dodecyl sulphate solution by measurement of sedimentation velocity, diffusion and effective partial specific volume. The polypeptide fraction exhibits a molecular weight of 25 000 which agrees with the apparent molecular weight found by polyacrylamide gel electrophoresis. The molecular weight of the polypeptidesodium dodecyl sulphate micelle was 54 000, with a friction ratio of 1.6 which indicates an effective asymmetric hydrodynamic shape. For binding measurements self-diffusion equilibrium dialysis with dodecyl [35S] sulphate was used. In this case, dialysis equilibrium was reached within about 10 hours, in contrast to the dialysis with initial concentration differences which requires much longer times. A binding value of δD = 1.15g sodium dodecyl sulphate per g polypeptide was obtained which corresponds to a molar binding ratio of 100 mol dodecyl sulphate bound per mol of polypeptide. After the removal of dodecyl sulphate the polypeptide is present in an aggregated state. In phosphate buffers of pH 6.8 and 7.5 the aggregates preponderantly have sedimentation coefficients of 11.7 and 6.8 Svedberg units respectively. Assuming equivalent spheres the molecular weights were calculated to be 340 000 and 150 000.

Purification and partial characterization of a rat kidney aldehyde dehydrogenase that oxidizes retinal to retinoic acid

1993 ◽  
Vol 71 (1-2) ◽  
pp. 85-89 ◽  
Author(s):  
Jean Labrecque ◽  
Pangala V. Bhat ◽  
André Lacroix
Keyword(s):  
Molecular Weight ◽  
Retinoic Acid ◽  
Vitamin A ◽  
Aldehyde Dehydrogenase ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Size Exclusion

A NAD-dependent aldehyde dehydrogenase (EC 1.2.1.3) which catalyzes the oxidation of retinal to retinoic acid was purified to homogeneity from rat kidney by using Affi-Gel blue affinity chromatography and chromatofocusing, followed by Mono-Q anion-exchange chromatography. The apparent molecular weight of the native enzyme determined by size-exclusion fast protein liquid chromatography was 140 000. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a subunit molecular weight of 53 000. The isoelectric point as measured by chromatofocusing was 8.5. The enzyme also catalyzed the oxidation of acetaldehyde, but showed much lower Km value for the retinal substrate. We suggest that aldehyde dehydrogenase found in the kidney may be a specific retinal dehydrogenase, involved in vitamin A metabolism.Key words: aldehyde dehydrogenase, vitamin A, retinal, retinoic acid, kidney.

scholarly journals Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes

1975 ◽  
Vol 151 (3) ◽  
pp. 685-697 ◽  
Author(s):  
M Letarte-Muirhead ◽  
A N Barclay ◽  
A F Williams
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Surface Glycoprotein ◽  
Cell Surface Glycoprotein ◽  
Lentil Lectin ◽  
Polypeptide Chains

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.

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.

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.

Factors Influencing Thrombin Digestion Of Human Platelet Thrombospondin

1981 ◽  
Author(s):  
J W Lawler ◽  
F C Chao ◽  
J Palek
Keyword(s):  
Molecular Weight ◽  
Disulfide Bonds ◽  
Human Platelet ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Platelet Protein ◽  
Flow Through ◽  
Proteolytic Cleavages ◽  
Human Blood Platelets

Exposure of human blood platelets to thrombin results in the rapid release of a 420,000 dalton glycoprotein, designated thrombospondin, which is comprised of three polypeptides of equivalent molecular weight which are cross- linked by disulfide bonds. When purified human platelet thrombospondin is exposed to thrombin (4 units/ml) or plasmin (2 units/ml) for prolonged periods of time, proteolytic cleavages occur which result in the removal of 10,000 dalton and 30,000 dalton polypeptides with a concomitant decrease in the apparent molecular weight of the thrombospondin chains (Lawler, J.W. and Slayter, H.S., submitted for publication). In contrast, when the supernatant from thrombin- treated platelets is exposed to additional thrombin (4 units/ml) the 30,000 dalton, but not the 10,000 dalton fragment is released from thrombospondin. Proteolytic release of the 10,000 dalton polypeptide was inhibited by calcium and a nondialyzable component of the supernatant preparations. To further characterize the nondialyzable component, the supernatant was subjected to heparin-Sepharose affinity chromatography. Stepwise elution with varying NaCl concentrations yields a non-affinity flow through peak, a low affinity peak (eluted with 0.45 M NaCl) and a high affinity peak (eluted with 2.0 M NaCl). In addition to thrombospondin, the low affinity peak contains a β-thromboglobulin like protein as judged by RIA, apparent molecular weight on SDS-polyacrylamide gel electrophoresis (7,500-9,000 dalton) and heparin affinity. After dialysis of the low affinity peak against 0.14 M NaCl, 15 mM Tris-HCl (pH 7.6), 2 mM CaCl2 containing 0.02% NaN3, thrombin treatment resulted in the proteolytic release of the 30,000 dalton polypeptide, but not the 10,000 dalton polypeptide, from thrombospondin. These results suggest that calcium and a low molecular weight platelet protein can affect the pattern of thrombin digestion of thrombospondin.

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