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.

Identification of Proteolytic Activities in the Cytosolic Compartment of Mature Human Erythrocytes

“…Thus, the presence of three acidic endopeptidases in the soluble fraction of human erythrocytes is established. As a whole, the properties of these cytosolic acid proteinases and especially their molecular sizes, the substrate specificity and the susceptibility to inhibitors (see below), are clearly reminiscent of the picture previously obtained following gel chromatography through the same column of the solubilized membranes from human erythrocytes [7,8]. Accordingly, the same nomenclature as already used for the three acidic membrane proteinases is herein adopted for the corresponding cytosolic enzymes, i.e.…”

“…Erythrocytes were completely deprived of leukocytes and platelets according to the previously described filtration technique [7] and lysed by three repeated cycles of freezing and thawing. The 'soluble fraction' was obtained by centrifugation of the lysate at lOOOOOxg for 1 h at 5°C.…”

“…Since then, several conflicting reports on this subject have appeared [2-51, mostly dealing with proteinase activities associated with the erythrocyte membrane [6]. The present state of the relevant research seems to indicate that three distinct acidic proteinases [7,8] are endowed in the membrane of mature erythrocytes.On the contrary, only few data are available concerning proteolytic activities in the soluble fraction of mature erythrocytes. Cytosolic erythrocyte arylamidases were described by Makinen and Makinen in 1971 [9] and two such aminopeptidases, specific for N-terminal arginine or lysine residues, have been recently purified and characterized by the same authors [lo].…”

“…Since then, several conflicting reports on this subject have appeared [2-51, mostly dealing with proteinase activities associated with the erythrocyte membrane [6]. The present state of the relevant research seems to indicate that three distinct acidic proteinases [7,8] are endowed in the membrane of mature erythrocytes.…”

“…This procedure allowed a number of proteinases to be identified in the erythrocyte cytosol while providing a tool for their selective though partial separation. The following peptidases were found to be present in the soluble fraction of mature human erythrocytes : (a) a neutral endopeptidase having an approximate molecular weight of 110000; (b) three acidic endopeptidases, with pH optima between 2.5 and 3.5, showing molecular and functional properties almost identical with those of the three proteinases previously purified from solubilized erythrocyte membranes [Pontremoli et al (1979) Biochem. J.…”