Cathepsins are a group of intracellular proteolytic enzymes located within the lysosomes. On the basis of the specificity towards their substrates, the cathepsins have been assigned the names: Cathepsins A, B, C, D, E, G and L.

Cathepsin A is widely distributed in animal tissues. It has been isolated from bovine spleen, chicken muscle and rat kidney lysosomes (Shibko, S. and A.L. Tappel, Biochem J., 95, 731, 1965). The enzyme is an endopeptidase with pH optimum at pH 5.5 Cathepsin A and Cathepsin D possess a great degree of similarity in their enzymatic behavior.

Cathepsin B (EC 3.4.22.1) is a sulfhydryl-dependent enzyme which is found in many tissues but bovine spleen is a rich source (Greenbaum, L.M. and J.S. Fruton, J. Biol Chem, 226, 173, 1957). This enzyme hydrolyzes substrates which are similar to those that are attacked by pancreatic trypsin, but the pH of optimal activity for Cathepsin B is in the range of pH 3-4.

Cathepsin C (EC 3.4.14.1) is also known as dipeptidyl transferase and has been purified from fresh bovine spleen (Metrione, R.M., Neves, A.G. and J.S. Fruton, Biochemistry, 5, 1597, 1966). It has also been isolated from rat liver and spleen. Substrates most susceptible to enzymatic hydrolysis by Cathepsin C are dipeptidyl amides or esters bearing a free a-NH2 group in the NH2- terminal position. Optimum activity is exhibited over the range of pH 5-6.

Cathepsin D (EC 3.4.23.5) has been found to be widely distributed in various tissues but spleen is a rich source of this enzyme. Cathepsins A and D work synergistically to hydrolyze protein substrates. The rabbit liver Cathepsin D has a molecular weight of approximately 58,000 and it can be stored at 4° at pH 3-4 or at pH 8 for days without loss of significant amount of its activity (Press, E.M., Porter, R.R. and J. Cebra, Biochem J., 74, 501, 1960). Cathepsin D acts as a true proteinase on substrates such as denatured hemoglobin at the pH range of 2.8-3.0. In this respect it behaves enzymatically similar to pepsin.

Cathepsin E is present in very small amounts in spleen, but bone marrow is a rich source of this enzyme. The enzyme has a pH optimum of 2.5. It shows no hydrolytic activity toward the substrates of Cathepsin A, B or C. Cathepsin G (EC 3.4.21.20) has been isolated from the lysosomes of polymorphonuclear leukocytes. It exhibits hydrolytic activity similar to chymotrypsin.

Cathepsin L (EC 3.4.22.15) has been isolated from rat liver lysosomes.

If assayed as a proteinase, a unit of catheptic activity is defined as the amount of enzyme which causes an increase of 0.001 Unit extinction/minute of digestion of the substrate at 37°C.

If assayed as an amidase, a unit of amidase activity is defined as that amount of enzyme which produces a 1% hydrolysis of substrate Amide/minute at 37°C. This same unit definition is used if the esterase or hydrolase procedure are employed to assay cathepsin activity.

If assayed as a transferase, a unit of catheptic activity is defined as the amount of enzyme required to catalyze the formation of 1 mole of hydroxamic acid/minute at 37°C.

The pH optimum used in defining a unit of enzyme activity will be determined by the type of cathepsin being assayed.

As mentioned above, a number of methods have been employed for assaying cathepsin activity. The method of choice will be influenced by the type of cathepsin under investigation. The detailed procedures for assaying cathepsin activities by various methods have been described in Methods in Enzymology (Colowick, S.P. and Kaplan, N.O.; Eds. VOL XIX, 285-315, 1970, Academic Press, New York).