Cathepsin rabbit liver

Quite recently, additional thiol proteinases have been found in lyso-somes. Cathepsin T, which catalyzed conversion of multiple forms of tyrosine aminotransferase, was demonstrated by Gk>hda and Pitot (20, 21). This enzyme does not cleave benzoylarginine-2-naphthylamide, a substrate for cathepsin B and cathepsin H. Melloni et al. (22, 23) reported the presence of a thiol peptidyl peptidase in a lysosomal membrane fraction of rabbit liver. In addition, cathepsin N with strong collagenolytic activity was found in bovine spleen and human placenta, although its lysosomal origin has not yet been demonstrated (24-26). 

Melloni et al. (22, 23) obtained three distinct enzymes from rabbit liver lysosomes that catalyze limited proteolysis of rabbit liver fructose-1,6-bisphosphatase, converting the neutral form to a form with an alkaline pH optimum. One of these proteinases (Mr = 70,000) is associated with the lysosomal membrane fraction. Since cathepsin B, H, and L are all present in the soluble fraction of lysosomes 61), this enzyme seems to be a new thiol proteinase of lysosomes. The enzyme is activated by cysteine, but not inhibited by leupeptin. Since it has only... 

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,... 

A, rat liver cathepsin D (43,000 mU/mg protein) A, rat TDL high-speed extract (103 mU/mg protein) , rat TDL H-enzyme (927 mU/mg protein) q, rabbit liver high-speed extract (18.4 mU/mg protein) and , rabbit spleen high-speed... 

Inactivation of Rabbit Liver and Muscle Aldolases by Limited Proteolysis by Lysosomal Cathepsin M... 

During the course of our earlier studies on the conversion of fructose 1,6-bisphosphatase (Fru-Pgase) from the neutral to the alkaline form by lysosomal proteinases from rabbit liver (14-16) we observed that a considerable fraction of the converting enzyme activity, designated CE-II (15), remained associated with the insoluble membrane fraction after disruption of the lysosomes by freezing and thawing. This membrane-bound activity was found to be almost fully expressed by undisrupted rabbit liver lysosomes (15, 17). Rabbit liver cathepsin B, which also exhibited Fru-Paase-converting enzyme activity (15, 17, 18), was not present in the membrane fraction and was not responsible for the activity observed with intact lysosomes (17). 

Fig. 1. Inactivation of rabbit liver aldolase by lysosomal proteinases. The broad peak of activity observed with the soluble fraction is due to the presence of at least four proteinases Fru-P2ase converting enzyme (CE-II), eathepsins B and L (CE-III), and cathepsin M. Cathepsin M also catalyzes the loss of Fru-P2ase activity measured at pH 7.5 (inactivation of Fru-P2ase). Only cathepsin M and CE-II were detected in the membrane fraction. Reprinted with permission from Pontremoli et al. (19).

Undisrupted rabbit liver lysosomes also catalyze the hydrolysis of substrates for leucine aminopeptidase and hexoseaminidase, but they do not hydrolyze benzyol-L-arginine- ff-naphthylamide (BANA), the classical substrate for cathepsin B, nor do they show any activity with substrates for 8-glucosidase (17). Thus the activities expressed by undisrupted lysosomes are restricted to those that are associated with the lysosomal membranes. The fact that the intralysosomal activities remain fully cryptic (e.g., for cathepsin B substrates) excludes the presence of disrupted or permeable lysosomes that might have accounted for the activities expressed by these lysosomal preparations. 

Effect of Treatment of Undisrupted Rabbit Liver Lysosomes with Trypsin ON Activities of CE-II and Cathepsin M... 

Cathepsin M activity was assayed with rabbit liver aldolase as the substrate. 

Also in support of the location of cathepsin M on the outer lysosomal surface were results obtained with a monoclonal antibody prepared against the rabbit liver enzyme. This antibody, but not another monoclonal antibody specific for platelet surface markers, could be shown to bind to the intact lysosomes. In addition, rabbit liver lysosomes could be shown to bind to monoclonal anti-cathepsin M coupled to Sepharose 4B, but not to Sepharose 4B alone or to a coupled monoclonal antibody directed against surface glycoprotein of human platelets (Table II). 

The amino acid composition of rabbit liver cathepsin B was similar to those reported previously for the enzymes from rat (23,24), mouse (25), and human (26) liver (Table III). The amino acid compositions of rabbit liver cathepsins M and B were similar except for higher contents of serine, histidine, cysteine, and tryptophan in the former. The molecular weights, based on the results of SDS gel electrophoresis, are also similar for cathepsin M and cathepsin B. The enzymes from rabbit liver are both glycoproteins, containing 2-3 equivalents each of mannose and glucosamine, the latter presumably present as N-acetylglucosamine (unpublished observations). 

Fig. 4. SDS-polyacrylamide slab-gel electrophoresis of cathepsins M (lane 2) and B (lane 3) purified from rabbit liver. Molecular weight standards are shown in lane 1 (S. Erickson-Viitanen et al, unpublished).

On the other hand, a number of native proteins tested were found to be better substrates for cathepsin M than for cathepsin B (Table IV). Muscle and liver aldolases were inactivated by cathepsins M and B at comparable rates (Table IV) but yeast glucose-6-phosphate dehydrogenase was inactivated much more rapidly by cathepsin M and neither rabbit liver pyruvate kinase nor rabbit muscle glyceraldehyde-3-phosphate dehydrogenase was inactivated on incubation with cathepsin B. 

Rabbit liver cathepsin M causes the inactivation of both muscle and liver aldolases by removing small peptides from the COOH-termini (Fig. 5). This is the same region that had previously been shown to be sensitive to digestion by carboxypeptidase, subtilisin, and human cathepsin B (28, see also Section I). However in contrast to cathepsin B, which acts pri-... 

Cathepsin H, which acts as an endopeptidase as well as an aminopeptidase, has been purified from rat liver (9), human liver 12), rat skin 13, 14), and rabbit lung 15, 16). The enzymes from rabbit lung and rat skin have been called a-AT-Bz-Arg-/3-naphthylamide hydrolase, but because of their actions as endoaminopeptidases and their other similarities to the liver enzyme, they may be designated as cathepsins H. Our recent results 40) obtained with antiserum raised in rabbits against rat liver cathepsin H indicate the presence of this enzyme in many organs and tissues of rats. 

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. 

Kominami and Katunuma (40) raised antisera to rat liver cathepsin B and H in rabbits, and examined the cross reactivities of cathepsin B,... 

Mammalian liver and muscle frucose bisphosphate aldolases are also very susceptible to limited proteolysis (5,53-55). Cathepsin B, cathepsin L, and papain catalyze the limited proteolysis of rabbit muscle and rat liver aldolases 50,51). In fact, decrease of aldolase activity in liver is observed during starvation 109) and after administration of lysosome-tropic agents 103). Leupeptin caused an increase in osmotic sensitivity of lysosomes and an increase in the activities of free lysosomal proteinases, such as cathepsin A and cathepsin D, and a moderate increase of cathepsin B and L, and resulted in a decrease in aldolase activity. The molecular properties of aldolase isolated from the livers of control rats and leupeptin-treated rats indicated that the decrease of aldolase activity is attributable to hydrolysis of a peptide linkage(s) near the carboxyterminal of the enzyme. However, care is necessary in determining whether proteolytic modification of enzymes... 

Inhibition by antiserum. An intracellular localization for cathepsin D different from that of the other lysosomal acid hydrolases in rat TDL led us to explore some of the biochemical properties of this enzyme. As illustrated in Figure 3, an antiserum prepared in rabbits against rat liver soluble lysosomal enzymes effectively inhibited rat liver cathepsin D, although it did not inhibit the cathepsin D of rat TDL. In this case the incubations were carried out at pH 5 instead of pH 3.6 to avoid dissociation of the antigen-antibody complex. Both rat liver and rat TDL cathepsin D, however, have identical pH activity curves toward denatured bovine hemoglobin as substrate. 

Figure 3. Effect of globulins from sera of control and of immunized rabbits on cathepsin D activity in high-speed extracts of rat liver and of rat TDL. High-speed extracts of rat liver and of rat TDL were preincubated with control globulins from unimmunized rabbits (o) or globulins from antisera of rabbits immunized against soluble rat liver lysosomal enzymes ( ) for 1 hr at 370. Enzyme activity was measured at pH 5.0 according to the description of Yago and Bowers (5). Reproduced with permission from the Journal of Biological Chemistry.

Fig. 5. Peptide bonds in rabbit muscle and liver aldolases hydrolyzed by cathepsin M. The COOH-terminal sequences of both enzymes are shown. The primary sites of cleavage are shown by the large arrows and the secondary sites by the small arrows. With muscie aldolase, additional peptides derived from the last 20 amino acid residues, but not including proline 342, are recovered (unpublished observation).

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