Identification of the aspartic proteinases from human erythrocyte membranes and gastric mucosa (slow-moving proteinase) as catalytically equivalent to cathepsin E

Abstract

Three aspartic proteinases with similar Mr values (approx. 80,000) but from distinct sources (human gastric mucosa, human erythrocyte membranes and rat spleen) were shown to have immunological cross-reactivity and comparable mobilities when subjected to polyacrylamide-gel electrophoresis under non-denaturing conditions. Kinetic parameters (kcat, Km and Ki) were determined for the interactions of the three enzymes with two synthetic chromogenic substrates and five inhibitors (naturally occurring and synthetic). On this basis it would appear that all of the enzymes should be considered equivalent to cathepsin E. pH-activity measurements indicated that the aspartic proteinase that originated from the erythrocyte membranes retained activity at a higher pH value than either of its readily soluble counterparts.

Human cathepsin E produced in E. coli

“…Values for k~a, were derived from Vma X = k,.,, " [E] where the active concentration of individual preparations of enzyme was determined by active site titration against pepstatin as described previously [18]. The protein inhibitor from Ascaris lumbricoides and the synthetic inhibitor H-77 described previously [4,7,17], were kindly provided by Dr. R.J. Peanasky, University of South Dakota, USA and Dr. P.A. Charlton (formerly) of Glaxo, Greenford, UK, respectively.…”

“…Proteolytic activity was measured against haemoglobin as substrate [14] in 0.17 M sodium citrate buffer, pH 3.1, when crude preparations needed to be evaluated; or utilising peptide substrates containing p-nitrophenylalanine (Nph) in the P~ position as a chromogenic reporter group [17] when purer enzyme samples were available. Kinetic analyses with two of these peptide substrates (generously provided by Dr. Ben M. Dunn, University of Florida, USA) were performed as described previously [4] except that the pH used was pH 3.1 at a final ionic strength of 0.1 M. Initial velocities were measured with at least six concentrations of each peptide substrate within an appropriate range in order to derive the kinetic constants, K, I1 and Vm~x. The estimated error for all measurements was always < 15%.…”

“…Pepsin and gastricsin are both present in the stomach while the latter is also secreted from the prostate into seminal fluid [1,2]; renin is produced by a number of cells and tissues [3] while cathepsin D is a ubiquitous enzyme found in the lysosomes of most cells. The most recently characterised enzyme is cathepsin E [4], which has been implicated in the biogenesis of the vasoconstrictor peptide, endothelin [5] and to have a possible role in enabling infection of cells by the human immunodeficiency virus [6]. Recently, it has also been identified, for the first time unequivocally, to be the enzyme responsible for processing of a defined antigen (ovalbumin) in a defined lymphocyte system [7].…”

Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxidized insulin

“…The optimum pH ofcathepsin E towards hemoglobin is 3-3.5, so its proteolytic activity and substrate speci- ficity were investigated previously only at acidic pHs [3,[12][13][14]. As far as cathepsin E can be active only at acidic pHs, its physiological roles in vivo would be highly restricted.…”

Analysis of Binding Interactions of Pepsin Inhibitor-3 To Mammalian and Malarial Aspartic Proteases

“…For this study, we have analyzed the inhibition of aspartic proteases by a 17 kD inhibitor known as pepsin inhibitor-3 (PI3), which was originally isolated from the nematode Ascaris suum that infects pigs and is closely related to A. lumbricoides that infects humans (21). Previous studies have found that PI3 is a tight binding inhibitor of pepsin, gastricsin (21), and cathepsin E (22)(23)(24).…”