Effects of Thrombin, Chymotrypsin and Aggregated Gamma-Globulins on the Proteins of the Human Platelet Membrane

1977 ◽  
Vol 37 (03) ◽  
pp. 396-406 ◽  
Author(s):  
B Podolsak
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Membrane Component ◽  
Before And After

SummaryAnalysis of platelet membrane proteins and glycoproteins by SDS Polyacrylamide gel electrophoresis was carried out before and after treatment with thrombin. Extended incubation with thrombin (in the presence of EDTA or adenosine, which inhibit aggregation) produced extensive changes in the bands observed. With incubation times of a few minutes however, the changes were restricted to a glycopeptide, GP IV (approx. 90,000 Daltons) and one or two polypeptides of low molecular weight, in particular polypeptide 16 (approx. 23,000 Daltons). At 0–3° C only polypeptide 16 was still hydrolyzed.Chymotrypsin, which does not activate platelets, attacked glycopeptides I, II, III but no changes were apparent in GP IV and polypeptide 16. When chymotrypsin-treated platelets were further incubated with thrombin, only GP IV and one to two low molecular weight polypeptides, especially polypeptide 16, were affected. As polypeptide 16 appears to be an integral membrane component it is possible that it, either by itself or in combination with GP IV, represents the primary thrombin substrate involved in platelet activation.Aggregated IgG, which also activates platelets, does not modify the membrane glycoproteins but does change the low molecular weight region in particular band 16.

Tryptic Peptide Maps Of The Major Human Blood Platelet Membrane Glycoproteins, Ia, IIa, IIb, IIIa, IIIb and IIIc

1981 ◽  
Author(s):  
J L McGregor ◽  
K J Clemetson ◽  
E James ◽  
P Clezardin ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Tryptic Peptide ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Blue Staining ◽  
Membrane Glycoproteins ◽  
Lectin Affinity Chromatography ◽  
Reducing Conditions

Some major platelet membrane glycoproteins separated by 2-dimensional polyacrylamide gel electrophoresis (isoelectric focusing, discontinuous SDS-gel electrophoresis) have been characterized by 2-dimensional tryptic peptide mapping. Human platelets were isolated, washed and surface-labelled by lactoperoxidase-catalyzed iodination. Labelled platelets were solubilized in SDS and separated by 2-dimensional gel electrophoresis under non-reducing conditions in the 1st dimension and either reducing or non-reducing conditions in the 2nd dimension. Alternatively, labelled platelets were solubilized in sodium deoxy- cholate (1 %) and the glycosylated components were isolated by lectin affinity-chromatography on Lens culinaris lectin or concanavalin A lectin and separated by 2-dimensional gel electrophoresis. The glycoproteins were cut out from 6 % polyacrylamide gels, after being identified with Coomassie blue staining and indirect autoradiography by their pI and molecular weight. Tryptic maps were prepared according to the method of Elder et al. The tryptic maps of GPIa, IIa, IIb, IIIa, IIIb and IIIc are different, with each showing a characteristic pattern with the possible exception of Ia and IIb which showed certain similarities in both the reduced and non-reduced states. GPIIa which is clearly separated under non-reducing conditions, appears from its tryptic map to be present in the Ib region when reduced. Thus this technique clearly identifies each GP by a parameter in addition to pI and molecular weight.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

Normal Mature Human Enamel Protein

1979 ◽  
Vol 58 (2_suppl) ◽  
pp. 986-987 ◽  
Author(s):  
A. Belcourt
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Organic Matrix ◽  
Low Molecular Weight ◽  
Human Enamel ◽  
Weak Density ◽  
Enamel Protein

Pure enamel was prepared using an original microdissection technic. Protein concentration was 375 μg per gram of enamel. Polyacrylamide gel electrophoresis showed a single fast-migrating zone containing a thin double band. Ultracentrifugation studies suggested that the proteins were of low molecular weight or of weak density. Absorption spectra showed a strong absorbance at 260nm. Amino acid analyses yielded a composition of 25% Gly, 13.5% Glu, 11% Ser, 11% Pro, 2% Cys and 2% Hyp. A glucidic content of 15% was estimated and glucose, galactose, mannose and fucose were identified. The organic matrix of enamel seemed to be constituted of two major glycoproteins probably fibrous but different from keratin.

scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

scholarly journals Serological grouping ofTreponema hyodysenteriae

1990 ◽  
Vol 105 (1) ◽  
pp. 79-85 ◽  
Author(s):  
D. J. Hampson ◽  
J. R. L. Mhoma ◽  
B. G. Combs ◽  
J. I. Lee
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Vaccine Development ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cross Reactivity ◽  
Low Molecular Weight ◽  
Serological Response ◽  
Double Immunodiffusion ◽  
Sds Page ◽  
Treponema Hyodysenteriae

SUMMARYTwo Australian isolates ofTreponema hyodysenteriaewhich did not fit within the current serological grouping system for these bacteria wrere examined by agarose gel double immunodiffusion tests (AGDP). Isolate Vic1 was serologically unique, and we propose that it becomes the type organism for a new sixth serological group ofT. hyodysenteriae(Group F). Isolate Q1 was unusual in that lipopolysaccharide (LPS) extracted from it reacted strongly in AGDP with serum raised against the type organism for serogroup D (A1), and also weakly with serum raised against the type organism for serogroup B (WA1). The nature of this cross-reactivity was examined by using cross-absorbed antisera in AGDP, and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.The pattern of serological cross-reactivity between Q1, A1 and WA1 was complex and was not fully defined, but the isolate Q1 apparently shared low molecular weight ‘serogroup’ LPS antigens with A1, and shared higher molecular weight LPS antigens with WA1. On this basis Q1 was designated as belonging to serogroup D, although it was recommended that this be qualified as D (B) to indicate the presence of weak cross-reactivity with serogroup B. Such serological cross-reactivity may have significance in relation to the development of immunity toT. hyodysenteriae. Isolate Q1 may be a potentially useful organism for vaccine development because of its ability to induce a good serological response to LPS of treponemes from both serogroups D and B.

Fractionation of Chromatin Components

1973 ◽  
Vol 51 (5) ◽  
pp. 709-720 ◽  
Author(s):  
John J. Monahan ◽  
Ross H. Hall
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Urea Solution ◽  
Equilibrium Density ◽  
Tissue Culture Cell ◽  
Low Molecular Weight ◽  
Nonhistone Proteins

A general method for isolation and fractionation of chromatin into its four major components, DNA, RNA, histories, and nonhistone proteins, is described. The procedure avoids the use of strongly acidic or alkaline conditions, or the use of ionic detergents or phenol. As few as 14 × 106 cells can be used. The procedure is reasonably rapid and has been used successfully with a number of tissue culture cell lines. The chromatin components are dissociated in a 3 M NaCl – 5 M urea solution containing 2-mercaptoethanol and EDTA. The DNA and high molecular weight RNA are collected by high-speed centrifugation and DNA is separated from the RNA by means of Cs2SO4 equilibrium density centrifugation. The histones, nonhistone proteins, and low molecular weight RNA's are fractionated using DEAE-cellulose column chromatography and polyacrylamide gel electrophoresis. A small amount (< 1%) of protein is present in the DNA and RNA fractions. At least 11 low molecular weight RNA subfractions can be detected by means of polyacrylamide gel electrophoresis.

scholarly journals Identification of the platelet-specific alloantigen, Naka, on platelet membrane glycoprotein IV

Blood ◽  
1990 ◽  
Vol 75 (3) ◽  
pp. 684-687 ◽  
Author(s):  
Y Tomiyama ◽  
H Take ◽  
H Ikeda ◽  
T Mitani ◽  
T Furubayashi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Platelet Membrane ◽  
Membrane Glycoprotein ◽  
Two Dimensional ◽  
Definitive Evidence ◽  
Sds Page ◽  
Platelet Transfusions

We describe the membrane localization of a new platelet-specific alloantigen, designated Naka, that is involved in refractoriness to HLA- matched platelet transfusions. By indirect immunoprecipitation, anti- Naka antibody precipitated a single, radiolabeled platelet membrane protein with a molecular weight (mol wt) of 91 Kd from Naka-positive platelets. When radiolabeled Naka-negative platelets were used as a source of target antigens, no radiolabeled proteins were precipitated. The analyses using nonreduced-reduced two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and using rabbit antiglycoprotein (GP)IV demonstrated that this protein corresponds to GPIV (alternatively GPIIIb). Furthermore, in dot immunobinding, anti- Naka antibody bound to purified GPIV. Our results provide definitive evidence that the Naka alloantigen is carried on GPIV. These results also demonstrate that, on occasion, antibodies against GPIV may play an important role in refractoriness to platelet transfusions.

Analysis of proteins in rabbit pulmonary surfactant using monoclonal antibodies

1986 ◽  
Vol 250 (3) ◽  
pp. C460-C467 ◽  
Author(s):  
R. J. King ◽  
H. M. Martin ◽  
J. B. Baseman ◽  
J. Morrison-Plummer
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
High Molecular Weight ◽  
Gel Electrophoresis ◽  
Pulmonary Surfactant ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Molecular Weight ◽  
Weight Group ◽  
Molecular Weights ◽  
Intermediate Group

We have used monoclonal antibodies developed against the apolipoproteins associated with pulmonary surfactant purified from rabbit lavage fluid to study the expression of epitopes common to these proteins. The pulmonary surfactant contained nearly 20 proteins, of which at least 10 were not derived from serum. Electrophoresis, with sulfhydryl reduction of these proteins indicated apparent molecular weights of approximately 155, 135, 125, and 115 X 10(3) (high-molecular-weight group); 80, 70, and 60 X 10(3) (intermediate group); and 18 through 10 X 10(3) (low-molecular-weight group). Two-dimensional polyacrylamide gel electrophoresis, in which the proteins were electrophoresed without reduction in the first dimension, but with sulfhydryl reduction in the second dimension, revealed that the 80, 70, and 60 X 10(3) proteins dissociated into proteins of nominal molecular weights of 40, 35, and 30 X 10(3), respectively. In contrast, the 125 and 115 X 10(3) proteins of the high-molecular-weight group contained a protein which could only be reduced to a minimum molecular weight of 55 to 60 X 10(3). Monoclonal antibodies generally were of three types: those that reacted strongly with the high-molecular-weight group and weakly with the intermediate group; those that reacted conversely; and those that reacted only with the low-molecular-weight group. Our results indicate that at least two different surfactant apolipoproteins, with differing minimum molecular weights in SDS-polyacrylamide gel electrophoresis, have common epitopes. Although these results cannot certify a physiological relationship between these proteins, they suggest that the intracellular synthesis or extracellular processing of surfactant apolipoproteins may be more complicated than predicted by the findings of previous experiments, perhaps involving the posttranslational assembly of one surfactant protein into oligomers which resist dissociation under the conditions used for the analyses.

Method for measuring the concentration of urinary proteins according to their molecular size category.

1976 ◽  
Vol 22 (5) ◽  
pp. 667-672 ◽  
Author(s):  
A J Pesce ◽  
A Hsu ◽  
C Kornhauser ◽  
K Sethi ◽  
B S Ooi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Low Molecular Weight ◽  
Tubular Proteinuria ◽  
Equal Concentration ◽  
Glomerular Proteinuria ◽  
Low Molecular Weight Proteins

Abstract We combined the use of a concentrating device (Minicon) and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to semi-quantitate the concentration of (a) the collective low-molecular-weight proteins and (b) of albumin excreted in the urine of patients after renal transplantation. Analytical recovery of many serum proteins from samples concentrated 100-fold in the Minicon apparatus was about 70%. It was possible to examine many urine samples by polyacrylamide gel electrophoresis after concentration with this device. The reproducibility (CV) of the technique was on the order of 20% when albumin and low-molecular-weight protein were in about equal concentration. The method was adequate to differntiate glomerular and tubular proteinuria, because in glomerular proteinuria the ratio of albumin to low-molecular-weight proteins is about 20/1, whereas in tubular proteinuria the ratio is about 1/1.

Sign in / Sign up

Export Citation Format

Share Document