David R. Phillips

The arrangement of proteins in the human erythrocyte membrane

Abstract When the iodination of intact human erythrocytes was catalyzed by the enzyme lactoperoxidase, only a single stroma protein was iodinated. This protein component thus appears to be the only protein component exposed on the exterior of the human erythrocyte surface. The apparent molecular weight of this membrane protein is 90,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Platelet plasma membrane glycoproteins Identification of a proteolytic substrate for thrombin

Abstract The surface glycoproteins of the platelet plasma membrane were labeled by oxidation with galactose oxidase followed by reduction with (3H)-sodium borohydride. Of the glycoproteins labeled, only glycoprotein V (apparent molecular weight of 89,000) was decreased as a result of thrombin action. The affected glycoprotein appeared to be completely removed at a concentration of 1 U thrombin per 109 platelets. A soluble glycopeptide hydrolytic product with an apparent molecular weight of 70,000 was released into solution.

Thrombin substrates and the proteolytic site of thrombin action on human-platelet plasma membranes

Abstract Intact or isolated plasma membranes of human platelets were treated with thrombin (EC 3.4.4.13), and the membrane proteins hydrolyzed by this enzyme were identified by acrylamide gel electrophoresis and lactoperoxidase-catalyzed iodination. In isolated membranes, a high-molecular-weight polypeptide (220 000) and three glycoproteins with molecular weights of 150 000, 118 000 and 93 000 were reduced in concentration and/or molecular weight after thrombin treatment. In intact membranes, however, only the 118 000-mol. wt glycoprotein was reduced in concentration. Although two other thrombin substrates (the 150 000- and 93 000-mol. wt glycoproteins) were exposed on the surface of intact membranes, they were not hydrolyzed. Thus, the plasma membrane of platelets appears to contain at least two thrombin substrates which because of their orientation in the membrane, are resistant to hydrolysis on intact platelets. The 118 000-mol. wt glycoprotein, the only protein hydrolyzed by thrombin on intact platelets, may be the proteolytic site of thrombin action on the membrane surface.

Position of glycoprotein polypeptide chain in the human erythrocyte membrane.

A study of the vectorial arrangement of the protein of human erythrocyte membrane showed that the 90,000 molecular weight class of protein was exposed on the ouside of the red cell membrane [1, 2]. This observation is based on the ability of the enzyme, lactoperoxidase, to catalyze the iodination of protein of this molecular weight class on the intact human erythrocyte. The analysis of the membrane protein separated on 5% acrylamide gels employed in these studies showed a glycoprotein present in the region of 90,000 molecular weight proteins. Recent studies have shown that glycoproreins have anomolous behavior on SDS disc gel electrophoresis systems [3, 4]. Cross-linking of the acrylamide gel in this system does not, however, affect the molecular weight estimation of protein. In order to extend our observations on the proteins that are iodinated by lactoperoxidase on the intact membrane, we have re-examined the membrane protein employing SDS electrophoresis on polyacrylamide gels with varying cross linkage. These studies have confirmed that a 90,000 molecular weight protein is exposed on the human erythrocyte membrane. In addition, a glycoprotein with an apparent molecular weight of 62,000 as determined by 10% disc gel electrophoresis is labeled and also occupies an exposed position on the membrane. 2. Materials and methods

An improved method for determining the actin filament content of nonmuscle cells by the dnase i inhibition assay

Abstract The actin filament content of cell lysates can be assayed by inhibition of DNase I activity (Blikstad, I., Markey, F., Carlsson, L., Persson, T., and Lindberg, U. (1978) Cell 15 , 935–943). We have modified this assay in two ways. We have (i) established lysis conditions that do not affect the state of polymerization of actin and (ii) used a computer for direct data acquisition and analysis of the linear portion of the reaction curve. These modifications allow the accurate determination of small changes in the actin filament content of intact cells.

Platelet plasma membrane lectin activity

Abstract The lectin activity of human platelet and erythrocyte membranes was evaluated using trypsinized, formalinized erythrocytes from eight species. Platelet membranes had the greatest lectin activity against cow erythrocytes, but also had significant activity against human, sheep, electric eel, and rabbit erythrocytes. In contrast, erythrocyte membranes only had low lectin activity against electric eel erythrocytes with no activity against the other types of erythrocytes tested. The platelet membrane lectin activity was found to reside in protein molecules on the external surface of the platelet plasma membrane. The lectin activity of platelet membranes was inhibited by amino sugars and some basic amino acids: N-acetylated amino sugars and other neutral sugars were without effect. These results demonstrate that the external surface of the platelet plasma membrane has a specific lectin activity.

Exterior proteins on the human erythrocyte membrane

Abstract Membranes from pronase treated human erythrocytes were isolated and the membrane proteins separated by disc gel electrophoresis in sodium dodecyl sulfate. A comparison of pronase treated cells and untreated cells show that pronase treatment of intact human erythrocytes results in the alteration of three membrane proteins with molecular weights of 90,000, 95,000, 105,000 and reduces their molecular size. All of the proteins with molecular weights of 90,000 and 105,000 were altered indicating that all proteins with these molecular weights must be exposed on the outside of the membrane. Protein fragments formed from proteolytic digestion remain with the membrane, and have an apparent molecular weight of 65,000. The residual fragments are insensitive to further hydrolysis, and appear to be portions of the hydrolyzed proteins which is buried within the membrane structure.

Expression of thrombin-enhanced platelet lectin activity is controlled by secretion

Recently,we demonstrated that thrombin-activated human platelets express a hemagglutination activity not expressed by non-activated platelets and that this activity mediates the direct platelet-platelet interactions which cause platelet aggregation [ 11. Although isolated membranes were found to contain the thrombinenhanced hemagglutinin [2], the control of its expression wasnot elucidated. In this study, we report that the expression of the agglutinin is secretiondependent, caused by secreted materials, and can express itself as both a platelet membrane-bound entity and a soluble agglutinin, and that the express sion of the soluble a~lut~~ is dependent on fibrin formation.

Cell Recognition Systems in Eukaryotic Cells

Direct interactions between cells are essential to the proper functioning of virtually all multicellular organisms. Study of these interactions extends to most disciplines in biology. In addition to bacterial adherence, the subject of this volume, excellent examples are to be found in embryology (interactions in developing tissues), hematology (hemostasis reactions), immunology (lymphocyte and macrophage interactions) and virology (viral infectivity). Although apparently unrelated, each of these processes depends on direct physical interactions between two or more membrane surfaces, and the ability of cells to discriminate between different membranes.