Lysosomal thiol proteinases

Previous studies have shown that muscle lysosomal hydrolases are released early in the postmortem period due to a decrease in intracellular ATP concentrations. The decreased intracellular ATP level causes the rupture of the lysosomal membrane (14), releasing hydrolytic enzymes (proteases, lipases, and glycosidases) that further potentiate the weakening of membrane integrity and cellular function. Furthermore, as the acidosis increases (due to the anaerobic conditions associated with cellular death) the intramuscular pH to levels reach that which are optimal for the activity of several lysosomal thiol proteinases. 

Duncan R, Cable HC, Lloyd JB, Rejmanova P, Kopecek J. Degradation of side-chains of N-(2-hydroxypropyl)methacry-lamide copolymers by lysosomal thiol-proteinases. Biosci Reps 1983 2 1041-1046. 

Cathepsin B is the best known and most thoroughly investigated lysosomal thiol proteinase. It has been isolated from many mammalian species and tissues, including liver (33-35), spleen (5, 6,35), preputial gland (36), lung (15,16), parathyroid gland (37), brain (38), and breast (39). It is probably present in all mammalian cells. Crystals of cathepsin B from rat liver are shown in Fig. 1 (19). 

All the lysosomal thiol proteinases described in Section II have been isolated only recently and few structural studies have been carried out. The amino acid composition of only cathepsin B has been reported and is quite similar to those of cathepsin B from human liver (75) and rat liver (19) however Ouchterlony double difPiision analysis with antiserum against rat liver cathepsin B showed no immunological cross reactivity (40). All the lysosomal enzymes examined were shown to contain carbohydrate. The 111 of Asn is a glycosylation site in rat liver cathepsin B (128) but no detailed analysis of this carbohydrate has been reported. Since human liver cathepsin H can be purified on Con A-Sepharose (12), it may also contain a carbohydrate moiety. Rat liver cathepsin H and L also bind to Con A-Sepharose. 

Proteinase inhibitors that inhibit cathepsin D and aminopeptidases slightly inhibit protein degradation in hepatocytes 63, 114) and cultured muscle 117). These findings also support the idea that lysosomal thiol proteinases play a major role in intracellular protein breakdown. In general, protein degradation is reduced by amounts of proteinase inhibitors that cause neither decrease of protein synthesis nor toxic effects 113). However, at higher concentrations or higher doses, they inhibit protein synthesis and have toxic effects 118). 

Addition of proteinase inhibitors to cells or their injection in vivo not only inhibits lysosomal proteinase activities but also causes formation of autophagic vacuoles 115) and induces synthesis of hemoglobinhydrolyzing thiol proteinase 16, 63). Increase of autophagic vacuoles in cells by treatment with leupeptin is responsible for inhibition of lysosomal thiol proteinase activities with consequent decrease of protein degradation. 

N-(2-Hydroxypropyl)methacrylamide copolymers bearing oligopeptide side chains with a terminal p-nitroaniline (NAp) were incubated with purified lysosomal enzymes (Tritosomes) in vitro, and cleavage of these chains was measured by monitoring the release of NAp. It was shown that certain sequences liberate the terminal residue on incubation at pH 5.5 with lysosomal enzymes Recently, the importance of lysosomal thiol-proteinases in N-(2-hydroxypropyl)methacrylamide copolymer side chain cleavage was discovered The synthesis of side-chain amino acid sequences chosen to match the known specificities of certain lysosomal thiol-proteinases resulted in a higher initial rate of NAp release and a greater extent of release (up to 50% of NAp bound for an incubation period of 5 h) Incubation of substrates in vitro... 

When it became apparent that the lysosomal thiol-proteinases were particularly important in the degradation of HPMA copolymer side-chains, a new series of side-chains was designed to meet known specificities of particular lysosomal enzymes.29 Two side-chains were synthesized (-Gly-Phe-Leu-Gly-Phe-NAp and -Gly-Gly-Phe-Leu-Gly-Phe-NAp) which contain a pentapeptide sequence previously shown O to be susceptible to the lysosomal proteinase cathepsin D, a thiol-independent enzyme. Five further sequences were prepared containing hydrophobic amino acids in the P2 and P positions in relation to the terminal bond (according to the terminology of... 

Schechter and Berger ). These side-chains were designed for cleavage by the lysosomal thiol-proteinase cathepsin L, a rat liver lysosomal enzyme 2 known to hydrolyse oxidized B-chain of insulin, such that hydrophobic residues are present in the 2 3... 

Structures and Functions of Lysosomal Thiol Proteinases and Their Endogenous Inhibitor... 

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