Dataset Information

Isolation and partial characterization of three acidic proteinases in erythrocyte membranes.

ABSTRACT: 1. The distribution of proteolytic activity in membranes from human erythrocytes and from rabbit reticulocytes and erythrocytes was investigated, after removal of leucocytes and platelets from the cell suspensions. 2. All membrane preparations displayed proteolytic activity in the acidic pH region only. Membranes from human and rabbit mature erythrocytes showed latent activity, which could be increased when extracted with a number of detergents. 3. Three active fractions were resolved either by gel chromatography of solubilized membrane extracts or by standard polyacrylamide-gel electrophoresis. The three proteinase activities (designated proteinases I, II and III) were purified from solubilized extracts of human erythrocyte membranes. 4. The relevant mol.wts. were around 80000, 40000 and 30000, respectively, and each of the three proteinases appeared to be composed of a single polypeptide chain. 5. Distinctive pH optima (in the range pH2.8-3.9) and different saturation profiles with globin as substrate were observed for proteinases I, II and III. 6. Dithioerythritol, Hg(2+) and Cu(2+) inhibited each of the three human enzymes, but more selective inhibitory effects were exerted by other modifiers of proteolytic enzymes and by haemin. Similar effects were observed with the three proteinases from rabbit cells. 7. The activity of the three human proteinases seems to be restricted to naturally occurring protein substrates, although with poor specificity, and none of them was active on synthetic substrates. 8. Digestion of globin by each of the three enzymes yielded similar polypeptide fragments in all cases, this indicating an endopeptidase type of activity.

SUBMITTER: Pontremoli S

PROVIDER: S-EPMC1161195 | biostudies-other | 1979 Sep

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or 'non-self' by the immune system and aggressively opsonized with IgG.

Dataset Information

Isolation and partial characterization of three acidic proteinases in erythrocyte membranes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets