Purification and Some Properties of Cathepsin A of Small Molecular Size from Pig Kidney

Abstract

Cathepsin A [EC 3.4.2.-] of small molecular size (cathepsin A, S) has been purified about 800-fold from pig kidney by procedures including chromatographies on DEAE-Sephadex, SP-Sephadex, and Sephadex G-150. 1. The homogeneity of the purified enzyme was proved by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight (100,000) and isoelectric point (pI=5.0) were estimated. 2. The enzyme was remarkably stabilized by sucrose and KCl, and was most stable at pH 5-5.5 in the presence of both stabilizers. The enzyme had not only peptidase activity but also esterase and amidase activity; it was optimally active at pH 5.2 for peptide hydrolysis and at pH 8 for the hydrolysis of esters and amides. 3. Diisopropyl fluorophosphate and iodoacetamide completely inhibited these three activities. 4. The enzyme hydrolyzed various benzoyl- and benzyloxycarbonyl-dipeptides with neutral, acidic, and basic amino acids, and proline in the C-terminal position. The carboxypeptidase nature of the enzyme was proved by its action on an oligopeptide. 5. Several enzymatic properties of cathepsin A, S were almost the same as thoas of cathepsin A of large molecular size (cathepsin A, L) and the crude homogenate.

Cathepsin A activity of a parietal thrombus of an abdominal aortic aneurysm

“…It splits C-end amino-acids from peptides and proteins, especially quickly if phenyloalanine residue occurs in the penultimate position, in a weakly acidic environment [12,[19][20][21]. Our results are in accord with this.…”

“…The whole 10% homogenate of thrombi and blood clots was prepared in 0.15 mol/l of KCl using a knife homogenizer, type Politron, and filtrated through polyamide fabric [11]. Z-Phe-Ala, Z-Phe-Phe, Z-Glu-Tyr, Z-Glu-Phe, Z--Gly-Phe, and Z-Gly-Ala, (Sigma-Aldrich, USA), were used to determine cathepsin A activity in homogenates [12,13]. Sodium-acetic acid buffer in the amount of 0.1 ml 0.2 mol/l was added to 0.3 ml of homogenate and, after adding 0.1 ml 30 mmol/l of the substrate dissolved in dimethyl sulfoxide, it was incubated at 37°C for four hours.…”

Dipeptidyl peptidase IV, a kidney brush-border serine peptidase

“…Cathepsin A, a lysosomal serine peptidase, has been described as existing in two forms with mol.wts. 500000 and 100000 (Kawamura et al, 1975), but it is not clear whether the latter represents the subunit size. The dimeric form of dipeptidyl peptidase IV in the isolated state is well established, but it is unknown if this is the form of the enzyme in the microvillus membrane.…”

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

“…During the last decade, cathepsin A has been shown to have both catalytic and protective functions. Cathepsin A has three distinctive hydrolytic activities for carboxypeptidase [23][24][25], deamidase and esterase [25 -28]. A deamidase that deamidates tachykinin peptides was recently purified from culture media of thrombin-stimulated human platelets and was subsequently identified as cathepsin A [24].…”