Purification of pancreatic carboxylic-ester hydrolase by immunoaffinity and its application to the human bile-salt-stimulated lipase.
A column of immobilized antibodies directed against pure human pancreatic carboxylic (cholesterol) ester hydrolase was used to purify in a single step the enzyme from human pancreatic juice as well as carboxylic-ester hydrolases from other species (rat, dog). This immunoaffinity method was also used for the purification of the related bile-salt-stimulated lipase from the human skim milk. The enzymes were homogeneous on SDS-PAGE. The yields obtained were always higher than those previously observed using either conventional or affinity columns. The human and dog carboxylic-ester hydrolases as well as the bile-salt-stimulated lipase, in contrast to the rat enzyme, are glycoproteins. From our results, it can be speculated that these enzymes, which differ in their molecular weight but not in their N-terminal sequences or amino-acid compositions, might have a similar proteic core with a molecular mass between 65 and 75 kDa. The difference in their respective molecular masses might result from a different level of glycosylation of pancreatic carboxylic-ester hydrolases (and milk bile-salt-stimulated lipase).
Abouakil
N
N
Centre de Biochimie et de Biologie Moléculaire du C.N.R.S., Marseille, France.
Rogalska
E
E
Bonicel
J
J
Lombardo
D
D
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001647:Bile Acids and Salts / Q000494:pharmacology*; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification*; D002642:Chick Embryo; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008049:Lipase / Q000302:isolation & purification*; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D010189:Pancreatic Juice / Q000201:enzymology; D013045:Species Specificity; D002787:Sterol Esterase / Q000302:isolation & purification*
D000596:Amino Acids; D001647:Bile Acids and Salts; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D002787:Sterol Esterase; D008049:Lipase