Quantification and gel electrophoretic analysis of proteins extracted from vascular endothelial cells.
The inner walls of tubular organs and body cavities are lined with a sheet composed of cells which control the passage through the walls and thus are of considerable physiological and biochemical interest. They are difficult to prepare but their content can be extracted and analysed, using laminar extraction and sensitive bioluminescence methods. This has been shown for the endothelial cells which line the interior of blood vessels. Although it is dealing with small amounts of material, only a minor part of it is consumed in the analyses. The bioluminescence measurements can thus be combined with other analytical procedures such as protein assay and electrophoresis. The possibilities of extending the biochemical information is of interest in the efforts to clarify the pathogenesis of vascular diseases. Laminar elution of protein from endothelial cells of the rat aorta was traced by the simultaneous efflux of adenine nucleotides. These were determined in a bioluminescence assay after conversion to ATP, while the fluorescamine reaction was used for the quantification of protein. The elution patterns obtained by these two methods showed an initial peak with a common maximum. The new possibilities of detecting relevant biochemical changes were evidenced by the finding of a marked protein loss in the fractions containing the outflux from the endothelial cells of diabetic rats. Electrophoresis of proteins eluted from endothelial cells resulted in separation into a large number of bands, but no differences were detected in the electropherograms at comparisons between normal and diabetic rats. It remains to be clarified, whether the protein depletion of the endothelial sheet is due to a concomitant loss of cells or to a cytoplasmatic loss without profound changes of the cytoplasmatic composition.
Department of Medical Cell Biology, University of Uppsala, Sweden.
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Brolin
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Naeser
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Hjertén
D000227:Adenine Nucleotides / Q000032:analysis; D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000503:physiopathology*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000032:analysis*; D008297:Male; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D051381:Rats
D000227:Adenine Nucleotides; D011506:Proteins