Cathepsins, kidney

Fig. 6.33. The structure of human calcitonin (compare with salmon calcitonin in Fig. 6.22). The descending arrows indicate sites of cleavage in rat liver lysosomal fraction (full arrows) and rat liver and kidney cytosolic fractions (broken arrows). The ascending arrows indicate sites of cleavage by cathepsin B1 (full arrows) and cathepsin D (broken arrows) [149].

Proteins papain/cathepsin-L protease fonily (Mar spicules of Tethya aurantia, Porif. from California Shimizu 1998 Land kidney of Mamm. and tropical fruits of Ang. MI). 

Elastase activity is not a universal property of proteolytic sulfhydryl-activated enzymes. There are abundant reports in the literature describing the disappearance of elastic fibers in vivo preceding the repair of damaged tissues, but there is no evidence as to how this is brought about. The tissue cathepsins, most of which are SH-activated, have received little systematic study, but Thomas and Partridge (1960) reported that cathepsins extracted from kidney and spleen by the method of De La Haba et al. (1955) did not digest elastin either in the presence or the absence of cysteine. 

Bones are constantly dissolved by osteoclasts and remineralized by osteoblasts in response to mechanical forces. Osteoclasts possess an acidic compartment and pass demineralized bone products to the periosteum (Sect. 1). They develop in stress-induced bony microcracks and are activated by differentiation factors secreted by osteoblasts, especially after menopause. Menopausal osteoporosis is controlled by drugs that are a stable form of pyrophosphate (bisphosphonate) or cathepsin K inhibitors (Sect. 2). The calcium ion concentration of blood is raised by parathyroid hormone and a vitamin D derivative called calcitriol. Parathyroid hormone causes kidneys to excrete phosphate, retain calcium, and activate calcitriol production (Sect. 3). Calcitriol induces calcium transporter proteins in osteoclasts and intestinal epithelium, where they move calcium from bone or diet into blood (Sect. 4). The chapter concludes with a discussion of calcitonin which lowers blood calcium concentrations by reversing parathyroid hormone effects on the kidney and inhibiting osteoclast activity (Sect. 5). 

Cathepsins. Intracellular proteinascs obtained Irom animal tissue extracts, the richest sources being liver, kidney and spleen. Located primarily in the lysosomal fraction within the cell. Part of the genera] enzymic apparatus of animal cells in most cases they do not specialize in functions characteristic of individual tissues. Review of cathepsins A -C J. S, Fruton in The Enzymes vol, 4, P. D. Boyer et at., Eds. (Academic Press, New York, I960) pp 233-241 of cathepsins A-E M. J. Mycek, Methods JEnzymol. 19, 285-315 (1970) of cathepsins B, D and G several authors, Res. Monogr. Celt Tissue Physiol. 2, 57-89, 181 -248 (1977) of cathepsins B, D, G, H, L, N and S several authors, Ciba Found. Symp. 75, 1-68 (1980). 

C34Hg3N509, Mr 685.90, A -acylated peptide isolated from culture broths of various streptomycetes (Strep-tomyces testaceus, S. argenteolus) needles, mp. 228-229 °C (decomp.). The substance acts as an inhibitor of aspartate proteinases (caiboxy proteinases), e. g.,pepsin, cathepsin D (a lysosomal protease), and is also active against renin, formed in excessive amounts in some forms of kidney disease. There are also the pepstatins B and C, containing an A(-hexanoyl or N- A-methylpentanoyl) group, respectively, in place of the AT-(3-methylbutanoyl) group. 

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. 

H, and L. Anti-cathepsin B and H sera quantitatively precipitated only the corresponding enzyme protein. Anti-cathepsin B serum reacted with cathepsin B, but not with cathepsin H or cathepsin L, and similarly anti-cathepsin H reacted only with cathepsin H, indicating that the three thiol proteinases are distinct. Immunological diffusions with antisera indicated that rat liver cathepsin B and H are immunologi-cally identical with cathepsin B and H from rat kidney, lung, spleen, brain, and heart. The immunological identity of cathepsin D from various tissues of rats was also demonstrated (60). 

When leupeptin and E-64 were injected in vivo, the activity of cathepsin B and L in the lysosomal fraction of liver was inhibited within 1 hour and the inhibition persisted for at leeist 6 hours, but gradually disappeared within 36 hours (64) (Fig. 4). There was no difference in the time courses of inhibition by most derivatives of E-64 and leupeptin tested, but some derivatives of E-64 were ineffective in vivo, although they inhibited cathepsin B and L in vitro. Inhibition of cathepsin B and L by injection of leupeptin or E-64 was as marked in the kidney as in the liver, but these compoimds were less effective in skeletal muscle and heart. Hashida et al. (72) showed that E-64 administered in vivo penetrates into lysosomes of the liver, possibly by permeation rather than by endocytosis. When H-labeled E-64 was injected into rats ip, high levels of radioactivity were observed in the serum after a short time and later in the cytosol fraction of liver. While radioactivity in the serum had already decreased 1 hour after the injection, that in the lysosomal fraction increased to a maximum after 6 hours and then gradually decreased (Fig. 5). E-64 was mostly present in the free form in the blood and the c3ftosol fraction but in protein-bound form in the lysosomal fraction. The time course and dose-response of inhibition of lysosomal cathepsin B activity by E-64 was closely correlated with the radioactivity in the protein-bound fraction of the lysosomes. 

Cathepsins. Cathepsins are intracellular proteases of animal origin. The occurrence of several such enzymes has been demonstrated in various tissues, including spleen, pituitary gland, kidney, thymus, etc. It is obvious that there is no reason to anticipate that all cathepsins will have similar properties to each other or to any other proteases. Cathepsins have been designated by both Roman numerals and by letters. Some of these enzymes have been identified with enzymes purified independently, as cathepsin III with leucine aminopeptidase. Several are activated by sulfhydryl compounds, some by metals. The isolation of the various cathepsins and studies of their substrate specificities are subjects currently under investigation, but because of the lower concentration of enzyme in the source materials and the number of related enzymes present, this area of investigation has not reached the development of the study of digestive enzymes. 

Still little is known about the system of intracellular peptidases. Some studies have been made of the so-called cathepsin, which is the intracellular peptidase system of manunalian kidney and spleen. The studies of Berg-mann and his collaborators have revealed that the system contains endopeptidases, carboxypeptidases and aminopeptidases. 

Tissue and species distribution. The existence of unique forms of cathepsin D in rat lymphoid tissues led us to examine other tissues and species for these enzymes. Rat liver cathepsin D is inhibited to exactly the same degree as that obtained from rat kidney, rat adrenal, rat fibroblast, human tonsils, bovine liver, bovine spleen, calf thymus, rabbit liver, and rabbit spleen (6,... 

In all the replacement reactions catalyzed by cathepsin and papain studied by Fruton and his collaborators the optimum pH of the replacement reaction was at 7-8, whereas that of hydrolysis is near to 5. The authors suggest that at normal physiological pH values the catalysis of replacement may represent a major intracellular function of cathepsins and papainases. But what measurements there are of intracellular pH in mammalian kidney, liver, and muscle indicate it to be nearer 6 than 7 (49,51,98). The pH dependence in these transfer reactions is related to the pK of the cation of the replacement agent. 

At least four proteolytic enzymes, known as kidney cathepsins I, II, III, and IV, were found in crude kidney extracts. In their substrate specificity these intracellular proteinases show similarities to pepsin, trypsin, aminop tidase, and carboxypeptidase, respectively (11). Plentl and Page (139) showed that cathepsins I and IV are not identical with renin. Schales, Holden, and Schales (160) differentiated renin from all four cathepsins. The experimental proof for the nonidentity of renin and kidney cathepsins is summarized in Table IV. 

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