Cathepsin D isozymes from porcine spleens. Large scale purification and polypeptide chain arrangements.
Six cathepsin D isozymes have been purified from porcine spleen using a large scale purification procedure. Five isozymes, I to V, have an identical molecular weight of 50,000 and are similar in specific activity. Isozymes I to IV contained two polypeptide chains each. The light and heavy chains have Mr = 15,000 and 35,000, respectively. Isozyme V is a single polypeptide. The molecular weight of the sixth isozyme is about 100,000 and it has only 5% of the specific activity of the other isozymes. On Ouchterlony immunodiffusion, an antiserum formed precipitin lines against the urea-denatured isozyme with Mr = 100,000. This immunoreactivity showed immunoidentity with those formed against other isozymes. The NH2-terminal sequence of light chains was identical for the isozymes. This sequence is homologous to the NH2-terminal sequence of other acid proteases, especially near the region of the active center aspartate-32. The NH2-terminal sequence of the single chain, isozyme V, Is apparently the same as the light chain sequence. The NH2-terminal sequence analysis of the heavy chain from isozyme I produced two sets of related sequences, suggesting the prescene of structural microheterogeneity. The carbohydrate analysis of the isozymes, the light chain, and the heavy chain revealed the presence of possibly four attachment sites, with one in the light chain and three in the heavy chain. Each carbohydrate unit contains 2 residues of mannose and 1 residue of glucosamine. The results suggest that the high molecular weight cathepsin D (Mr = 100,000) is the probable precursor of the single chain (Mr = 50,000), which in turn produces the two-chain isozymes. These are likely in vivo processes.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002403:Cathepsins / Q000302:isolation & purification*; D002417:Cattle; D012085:Chymosin; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010434:Pepsin A; D013045:Species Specificity; D013154:Spleen / Q000201:enzymology*; D013552:Swine
D000596:Amino Acids; D002241:Carbohydrates; D007527:Isoenzymes; D046911:Macromolecular Substances; D002403:Cathepsins; D010434:Pepsin A; D012085:Chymosin