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

Abstract

Individual lysates from human erythrocyte suspensions, completely deprived of leucocytes and platelets, were assayed for a number of proteolytic activities using both naturally occurring and synthetic substrates. Removal of hemoglobin by batchwise DEAE-cellulose chromatography did not modify the complement of the various proteolytic activities which were then fractionated by means of chromatography on a column of DEAE-cellulose, followed by conventional techniques such as gel chromatography and preparative electrophoresis. 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. 181, 559 -5681; (c) two dipeptidylaminopeptidases whose molecular weights are around 80000 and tentatively identified as dipeptidyl aminopeptidases I1 and 111, respectively, on the basis of their substrate specificities and pH optima; (d) presumably two aminopeptidases, having an approximate molecular weight of 80 000 and classified as an aminopeptidase with broad substrate specificity and an aminopeptidase B, respectively. No evidence for any carboxypeptidase activity was found in the cytosolic compartment of mature human erythrocytes.The occurrence of proteolytic enzymes in human erythrocytes was originally described in 1953 by Morrison and Neurath [l]. 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]. MoreAbbreviation. CI~ACOH, trichloroacetic acid. Enzymes. Aminopeptidase (microsomal) (EC 3.4.1 1.1); dipeptidylaminopeptidase I1 (EC 3.4.14.2); serine carboxypeptidase (EC 3.14.16.1).over, the presence of dipeptidylaminopeptidases, more specifically indicated as dipeptidylaminopeptidase 111, has been reported [ll]. Another soluble proteolytic system has been described, but in rabbit reticulocytes and not in mature erythrocytes. This enzyme, which is characterized by its energy dependence, has been postulated to be responsible for the degradation of structurally abnorma...

Extralysosomal Protein Degradation

“…In human (110)(111)(112) and porcine erythrocytes (113) only one type of CANP is present. The enzyme shows intermediate requirements of Ca2+ with KO.5 of 40-50 f-tM; thus it is closer in its properties to the mM fonn of CANP.…”

Site-directed activation of calpain is promoted by a membrane-associated natural activator protein

“…Although the association of calpain with membranes can also promote its autoproteolytic interconversion at low [Ca2+], the kinetics of this activation are not consistent with the actual rate measured in cells 16,7]. In human [8] and bovine brain [9], and more recently in human platelets [10], human neutrophils [11] and rat skeletal muscle [12], an endogenous calpain activator branes, but not to phospholipid vesicles, suggesting the participation of an intrinsic membrane protein(s).…”

Re-evaluation of the structural integrity of red-cell glycoproteins during aging in vivo and nutrient deprivation

“…Red-cell filtration inhibited the release by 54+ 30% (P < 0.05) from cells nutrient-deprived in Ca2+ and by 68 + 29% (P < 0.02) from cells kept in EDTA in all four experiments. Complexation of Ca2 , which is known to activate certain white-cell proteinases (Bocci et al, 1981;Legendre & Jones, 1983;Pontremoli et al, 1985), and erythrocyte calpain (Pontremoli et al, 1980) similarly inhibited sialoglycopeptide release by 77 + 33 (P < 0.02) for unfiltered red cells and by 86+13% (P < 0.01) for Fig. 2.…”