Protein Distribution in Human Endothelial Cells in Primary Culture Studied by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophores

1979 ◽  
Author(s):  
T. Børsum ◽  
I. Hagen ◽  
T. Henriksen ◽  
B. Carlander
Keyword(s):  
Endothelial Cells ◽  
Protein Pattern ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Major Protein ◽  
Protein Distribution ◽  
Human Endothelial Cells ◽  
Neuraminidase Treatment ◽  
Sds Page ◽  
Pas Staining

The surface structure and protein composition of cultured human endothelial cells were examined by lactoperoxidase catalyzed iodonation followed by SDS-PAGE of the cells. Five major protein bands (MW 224.000, 187.000, 70.000, 52.000 and 42.000) and several minor bands were seen. No differences in the protein pattern were observed between samples from confluent and nonconfluent cultures. In contrast, PAS staining of the gels revealed no glycoprotein bands unless the cells had grown to confluency. Then PAS staining revealed three bands of MW 240.000, 208.000 and 145.000. The fire one may represent fibronectin. The radioavtive iodine was associated with proteins in the 230.000, 145.000, 70.000, 55.000 and 45.000 MW regions. The first two regions showed iodination only in samples from confluent cultures. 33% of the acid-hydrolyzable sialic acid was liberated after neuraminidase treatment of the cells. No difference in the protein or glycoprotein pattern was seen after SDS-PAGE of neuraminidase-treated cells. In conclusion, four of the protein bands were susceptible to iodination, indicating an external localization in the plasma membrane. Two of these were iodinated only when cells had grown to confluency.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

scholarly journals Construction and Characterization of anagr Deletion Mutant of Staphylococcus epidermidis

2000 ◽  
Vol 68 (3) ◽  
pp. 1048-1053 ◽  
Author(s):  
Cuong Vuong ◽  
Friedrich Götz ◽  
Michael Otto
Keyword(s):  
Staphylococcus Epidermidis ◽  
Deletion Mutant ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Proteins ◽  
Accessory Gene ◽  
Accessory Gene Regulator ◽  
Sds Page ◽  
The Difference

ABSTRACT The physiological significance of the accessory gene regulator (agr) system of Staphylococcus epidermidis was investigated by construction of an agr deletion mutant via allelic replacement with a spectinomycin resistance cassette. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the protein pattern was strongly altered in the mutant; the amounts of most surface proteins were higher, whereas the amounts of most exoproteins were lower. The agrsystem of S. epidermidis thus appears to have an important impact on growth phase-dependent protein synthesis as has been shown for Staphylococcus aureus. The activity of the exoenzymes lipase and protease, assumed to be involved in staphylococcal pathogenicity, was investigated by agar diffusion assays and SDS-PAGE activity staining. A general reduction of these enzyme activities in the agr mutant was found. The difference in overall lipase activity was small, but zymographic analysis suggested a clear defect in lipase processing in the agr mutant.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

Abstract The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

EXPRESSION OF PLATELET ALLOANTIGENS ON HUMAN ENDOTHELIAL CELLS AND HEL CELLS

1987 ◽  
Author(s):  
Y Kawai ◽  
R R Montgomery ◽  
K Furihata ◽  
T J Kunicki
Keyword(s):  
Endothelial Cells ◽  
Protein A ◽  
Cell Types ◽  
Membrane Glycoproteins ◽  
Human Endothelial Cells ◽  
Immunoblot Assay ◽  
Sds Page ◽  
Hel Cells ◽  
Erythroleukemia Cell ◽  
Different Cell Types

Analogs of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa) have been shown to be synthesized and expressed by human endothelial cells (HEC), a human erythroleukemia cell line (HEL) and various other cells. Previous studies from our laboratory demonstrated that the platelet alloantigen P1A1, is expressed on HEC GPIIIa. Other alloantigen systems, namely, Pen and Bak, are known to be localized on platelet GPIIIa and GPIIb, respectively. Utilizing additional antibodies from patients with PTP specific for Pena, Baka, and Bakb allo-antigens, and isoantibodies (iso-ab) from a patient with Glanzmann's Thrombasthenia (GT), we have studied cultured HEC and HEL cells for expression of epitopes recognized by these antibodies. HEC and HEL cells were meta-bolically labeled with 35S-methionine and lysed in 0.5% TX-100 in the presence of 5mM EDTA. Soluble antigens were immunoprecipitated with these antibodies coupled to Protein A-Sepharose and subjected to SDS-PAGE and fluorography. Anti-Pena and the GT iso-ab reacted with the GPIIb-IIIa complex from both HEC and HEL lysates, but anti-Baka and anti-Bakb failed to immunoprecipitate GPIIb-IIIa analogs from either HEC or HEL. In an immunoblot assay, the GT iso-ab bound to GPIIIa of both HEC and HEL. Anti-Pena failed to react with SDS-denatured proteins. HEL GPIIIa migrates slightly faster than HEC GPIIIa and slightly slower than platelet GPIIIa. These results indicate that the epitopes of platelet GPIIIa recognized by alloantibodies and isoantibodies are shared by GPIIIa analogs of HEC and HEL. GPIIb-associated alloantigens are not expressed by HEC and HEL GPIIb analogs, an observation that is consistent with the decreased structural homology between GPIIb analogs derived from different cell types.

Changes in total and individual proteins during drying and ruminal fermentation of alfalfa

2005 ◽  
Vol 2005 ◽  
pp. 198-198
Author(s):  
A. A. Sadeghi ◽  
P. Shawrang ◽  
M. Moradi ◽  
A. Nikkhah
Keyword(s):  
Crude Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Plant Proteins ◽  
Ruminal Fermentation ◽  
Protein Nitrogen ◽  
Sds Page ◽  
Total Crude Protein ◽  
Hydrolysis Of

Proteolysis within plant cells occurs during wilting and drying. Changes in plant proteins during those periods usually are monitored by measurement of total crude protein and non protein nitrogen. Alternatively, changes in concentrations of individual proteins can be measured. Plants are composed of an array of different proteins. Electrophoresis can be used to separate these proteins and has been used to study effects of wilting and ensiling on proteins of some forages (Grum et al., 1991). Electrophoresis also has been used in the study of ruminal hydrolysis of oilseed meals proteins (Sadeghi et al., 2004). Most of the experiments designed to use electrophoresis to study protein metabolism in forages and ruminants have been qualitative. The main objective of this study was to determine whether sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry could be used to monitor quantitatively the changes in alfalfa protein composition during wilting, drying and ruminal exposure.

scholarly journals Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

1983 ◽  
Vol 96 (4) ◽  
pp. 1030-1039 ◽  
Author(s):  
W J Brown ◽  
W A Shannon ◽  
W J Snell
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Granule Protein ◽  
Isolation And Characterization ◽  
Cytoplasmic Face ◽  
Sds Page ◽  
Granule Membrane ◽  
Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

scholarly journals Synthesis of proteins from [35S]methionine by guinea pig megakaryocytes in vivo and time course of appearance of newly synthesized proteins in platelets

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 887-891 ◽  
Author(s):  
BP Schick
Keyword(s):  
Protein Synthesis ◽  
Guinea Pigs ◽  
Time Course ◽  
Protein Pattern ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Weights ◽  
Sds Page ◽  
Relationship Of

The relationship of protein synthesis to megakaryocyte maturation has been studied in guinea pigs in vivo. Guinea pigs were injected with a single dose of [35S]methionine. Megakaryocytes and platelets were isolated daily for 4 days, and proteins from both cells were isolated by DEAE-Sephacel chromatography and analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. All proteins in megakaryocytes corresponding to stained bands on the SDS- PAGE gels were radiolabeled at 3 hours after injection. The greatest loss of radioactivity from the megakaryocytes occurred between 1 and 3 days after injection. Only trace labeling of platelet proteins was seen at 3 hours, representing almost entirely three bands at molecular weights 47,000, 52,000, and 66,000. At 24 hours only about 13% of the maximal labeling was present, but not all proteins were labeled. The maximal labeling was at 3 days. The pattern of labeling of platelets at 3 days was identical to that of megakaryocytes at 3 hours. The protein pattern of nonmegakaryocytic marrow cells was different from that of the platelets and megakaryocytes. Data presented here suggest that most protein synthesis in megakaryocytes is completed at least 24 hours before release of the platelets to the circulation, and suggest some specificity in the proteins that are synthesized at the terminal stages of maturation.

scholarly journals High-molecular weight kininogen is present in cultured human endothelial cells: localization, isolation, and characterization

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1268-1276 ◽  
Author(s):  
F van Iwaarden ◽  
PG de Groot ◽  
JJ Sixma ◽  
M Berrettini ◽  
BN Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Endothelial Cells ◽  
Isolation And Characterization

The presence of high-molecular weight (mol wt) kininogen was demonstrated in cultured human endothelial cells derived from the umbilical cord by immunofluorescence techniques. Cultured human endothelial cells contain 58 +/- 11 ng (n = 16) high-mol wt kininogen/10(6) cells as determined by an enzyme-linked immunosorbent assay (ELISA) specific for high-mol wt kininogen. High-mol wt kininogen was isolated from cultured human endothelial cells by immunoaffinity chromatography. Nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that endothelial cell high-mol wt kininogen consisted of five protein bands with mol wts of 95,000, 85,000, 65,000, 46,000, and 30,000 daltons. Immunoblotting of the endothelial cell high-mol wt kininogen by using specific antisera against the heavy and light chain indicated that the 95,000-, 85,000-, and 65,000-dalton bands consisted of the heavy and light chain whereas the 46,000- and 30,000-dalton bands reacted only with the anti-light chain antiserum. Immunoprecipitation studies performed with lysed, metabolically labeled endothelial cells and monospecific antisera directed against high-mol wt kininogen suggested that high-mol wt kininogen is not synthesized by the endothelial cells. Endothelial cells cultured in high-mol wt kininogen-free medium did not contain high-mol wt kininogen. These studies indicate that endothelial cell high-mol wt kininogen was proteolytically cleaved in the culture medium and subsequently internalized by the endothelial cells. Binding and internalization studies performed with 125I-labeled, proteolytically cleaved, high-mol wt kininogen showed that endothelial cells can indeed bind and internalize proteolytically cleaved high-mol wt kininogen in a specific and saturable way.

scholarly journals Human vascular endothelial cells express a membrane protein complex immunochemically indistinguishable from the platelet VLA-2 (glycoprotein Ia-IIa) complex

Blood ◽  
1989 ◽  
Vol 73 (5) ◽  
pp. 1235-1241 ◽  
Author(s):  
JC Giltay ◽  
HJ Brinkman ◽  
PW Modderman ◽  
AE von dem Borne ◽  
JA van Mourik
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fibronectin Receptor ◽  
Vitronectin Receptor ◽  
Surface Molecules ◽  
Sds Page ◽  
Collagen Receptor

Abstract Endothelial cells express surface molecules that are involved in cell- matrix interaction, including the vitronectin receptor and the fibronectin receptor, both members of a family of cell adhesion receptors (integrins). Here we provide evidence that endothelial cells express a membrane molecule, indistinguishable from the platelet VLA-2 complex, which is a collagen receptor and a member of the integrin family. To identify this endothelial molecule, we have used a monoclonal antibody, CLB-10G11, which recognizes the VLA-2 complex from platelets. The molecule recognized by CLB-10G11 from endothelial cells was characterized as follows. (1) The monoclonal antibody precipitated two proteins from surface-labeled endothelial cells that corresponded to the platelet VLA-2 subunits (glycoprotein Ia and IIa) as judged by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional nonreduced/reduced SDS- PAGE. (2) Preclearing of endothelial cells with monoclonal antibody A- 1A5, an antibody that is directed against the common VLA beta subunit, removed all the CLB-10G11-binding material. (3) Crossed immunoelectrophoresis revealed that CLB-10G11 recognizes a single precipitation arc from either platelets or endothelial cells. Analysis of these two cell types in one gel again revealed one precipitation arc. The antigen of either cell type, recognized by CLB-10G11 could be precipitated by either polyclonal antiplatelet or polyclonal antiendothelial cell antiserum. Hence, it appears that endothelial cells express at least three different surface molecules (the vitronectin receptor, the fibronectin receptor and a collagen receptor), which may play an important role in controlling the anchorage of endothelial cells to the extracellular matrix.

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