Proteolysis of canine apolipoprotein by acid proteases in canine liver lysosomes.
Canine liver lysosomes were purified by sucrose discontinuous density gradient centrifugation and then ruptured by sonication to obtain the soluble fraction. This soluble lysosomal fraction, which contained a 25-fold increase in acid phosphatase activity per mg of total protein when compared with the original homogenate, was incubated with a subfraction (1.110 less than d less than 1.210 g/cm3, HDL3) of canine high density lipoproteins (HDL) at pH 3.8. HDL3 proteolysis by lysosomal proteases, measured as the release of peptides and amino acids by the ninhydrin reaction, followed hyperbolic curves with straight lines (r = 0.99) obtained on Lineweaver-Burk plots. Km calculated from the Lineweaver-Burk plot was 635 mug of HDL3 protein per 0.5 ml of incubation mixture. Optimum HDL3 proteolysis was observed from pH 3.8 to 4.5. Incubation with the other subcellular organelle fractions did not result in HDL3 proteolysis. To evaluate the effects of enzyme inhibitors, iodoacetate, p-chloromercuribenzoate (both specific for the endopeptidase, cathepsin B (EC 3.4.22.1)) and pepstatin (specific for the endopeptidase, cathepsin D (EC 3.4.23.5) were tested. Iodoacetate and p-chloromercuribenzoate inhibited HDL3 proteolysis 100% and bovine serum albumin proteolysis 65%. Pepstatin inhibited HDL3 proteolysis 45% and bovine serum albumin proteolysis 70%. The in vitro data presented support the hypothesis that hepatic lysosomes play an important role in HDL3 catabolism in the dog. Furthermore, results obtained from enzyme inhibition studies suggest that a specific lysosomal endopeptidase, cathepsin B, may play the key role in HDL3 proteolysis.
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D001059:Apoproteins / Q000097:blood*; D002458:Cell Fractionation; D004285:Dogs; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000378:metabolism
D001059:Apoproteins; D007461:Iodoacetates; D008075:Lipoproteins, HDL; D013385:Succinate Dehydrogenase; D000135:Acid Phosphatase; D005952:Glucose-6-Phosphatase; D010447:Peptide Hydrolases