Purification cathepsin

Barrett, A. J. Cathepsin D. Purification of isoenzymes from human and chicken liver. Biochem. J. 77 7, 601 (1970)... 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

Okitani, A., Matsukura, U., Kato, H., and Fujimaki, M. (1980). Purification and some properties of a myofibrilar protein-degrading protease, cathepsin L from rabbit skeletal muscle. J. Biochem. 87,1133 1143. 

Nakamura, K., S. Yonezawa and N. Yoshizaki. Vitellogenesis-related ovary cathepsin D from Xenopus laevis. purification and properties in comparison with liver cathepsin D. Comp. Biochem. Physiol. 113B 835-840, 1996. 

Sherman, I. W., and Tanigoshi, L. (1983a). Purification of Plasmodium lophurae cathepsin D and its effects on membrane proteins. Mol. Biochem. Parasitol. 8, 207-226. 

Smith, A. M., Dowd, A. J., McGonigle, S. et al. (1993) Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica. Mol. Biochem. Parasitol. 62 1-8. 

Dufour, E., Obled, A., Valin, C., Bechet, D., Ribadeaudumas, B. Huet, J.C. (1987). Purification and amino-acid sequence of chicken liver cathepsin-L. Biochemistry, 26, 5689-95. 

Wada, K. Tanabe, T. (1988). Purification and characterization of chicken liver cathepsin-B. J. Biochem., 104, 472-6. 

Separation of human amylase isoenzymes 274 Purification of cathepsin D from rat spleen 275... 

Purification of bull seminal plasma hyaluronidase Purification of ribonuclease B Purification of biliary glycoprotein I Separation of human amylase isoenzymes Purification of cathepsin D from rat spleen Purification of a cathepsin E-like acid proteinase from rat spleen... 

Distributions of Cathepsin B, H, and L in Various Species and Tissues, and Their Purifications... 

Reports of the isolation of cathepsin L from rat liver lysosomes appeared independently from the East German workers 8, 9) and from our laboratory 10,11). Tie Martino et al. 41) and Lynen et al. 42) also reported purification of this enzyme from rat liver. Purification of the enzyme from rabbit skeletal muscle 17) and rat kidney 18) has recently been reported. 

Affinity chromatography of bacterial 244 lactate dehydrogenases separation of D-and L-specific enzymes by alteration of elution conditions Purification of cathepsin D by 245... 

Two representative procedures are presented in detail. The first, preparation of Z-Gly-Leu-PheCH-Cl, involves deblocking of Z-PheCH-Cl and subsequent coupling of the deblocked chloromethyl ketone with Z-Gly-Leu-OH. This compound is an excellent inhibitor of chymotryp-sin and cathepsin G. The second procedure, that for Ac-Ala-Ala-Pro- ValCH-Cl, illustrates the synthesis of a peptide chloromethyl ketone from Boc-Val-OH with a minimum of isolation and purification along the way this compound is an excellent inhibitor of porcine pancreatic and human leukocyte elastase. ... 

At best, the evidence is indirect that cathepsins which toward peptides exhibit specificity patterns similar to those of known proteinases can, in fact, hydrolyze proteins. A satisfactory answer to this question must await the purification of the cathepsins to the degree of the extracellular proteinases. The main differences between the extracellular proteinases and cathepsins are as follows (1) The presence of a reducing agent is essential for the activity of cathepsins B and C but not for trypsin or chymotrypsin. (2) Crystalline tyrpsin inhibitor has no effect on cathepsin B. (3) The pH optima for cathepsins B and C lie between pH 3.5 and 6 compared to pH 8 to 9 favorable for the action of trypsin and chymotrypsin. 

Figure 1. Analytical Isoelectric Focusing at Various Stages in the Purification of Human Cathepsin D. (a) Homogenate,...

No change in the sensitivity of rat liver cathepsin D was noted during the course of a 200-fold purification, and thus it is unlikely that pepstatin-binding contaminants are present in the preparation (6). ... 

An interesting series of small peptides is represented by members such as Ala-Phe-NH2, His-Phe-NH2, Phe-Phe-NH2, and Phe-Phe. These peptides were all cleaved by cathepsin D preparations at purities of 50-100 units/mg protein, but when the purity rose to 150-200 units/mg these activities were lost. This digestion was obviously due to an impurity, possibly an aminopeptidase contaminant. The odd feature was that cleavage of these peptides was completely blocked by the addition of pepstatin or by the enzyme preparation s reaction with diazoacetylnorleucine methyl ester in the presence of cupric ions. These treatments also abolished all activity against the peptides listed in Table I. It must be concluded that the peptidase impurity had all the characteristics of a carboxyl type of enzyme. Possibly, some of the multiple forms of cathepsin D had a broader specificity than others, and these were lost as purification progressed. 

Oklahoma City, Large-Scale Purification of Cathepsin D from Porcine Spleen . 

---