A fully active, two-active-site, single-chain sucrase.isomaltase from pig small intestine. Implications for the biosynthesis of a mammalian integral stalked membrane protein.
Detergent-solubilized pig intestinal sucrase . isomaltase (EC 3.2.1.48-EC 3.2.1.10) was purified 40 to 100 times with a yield of 10 to 20% by a rapid immunoadsorbent technique. The purified enzyme was shown to be homogeneous by immunoelectrophoresis and was essentially free of other known brush border peptidases and disaccharidases. Intestinal sucrase . isomaltase isolated from pigs with intact pancreatic ducts consisted of two polypeptide chains with apparent molecular weights of 140,000 and 150,000, respectively. In contrast, the enzyme isolated from pigs in which the pancreas was completely disconnected from the duodenum 3 days before killing migrated in polyacrylamide gel electrophoresis in dodecyl sulfate as a single polypeptide chain with an apparent molecular weight of 260,000. Treatment with pancreatic proteases in vitro converted the large polypeptide chain into bands with molecular weights equal to or somewhat larger than those of sucrase . isomaltase purified from normal pigs. No increase of enzymatic activity could be detected during this transformation. It is suggested that the single-chain sucrase . isomaltase represents a precursor, which is converted to the final sucrase . isomaltase in vivo by pancreatic proteolytic enzymes. This is one of the few examples in vertebrates of a single polypeptide chain carrying two enzymatically active sites. The significance of the result for the mechanism of the biosynthesis of sucrase . isomaltase is discussed.
D000818:Animals; D010450:Endopeptidases; D007421:Intestine, Small / Q000201:enzymology*; D008565:Membrane Proteins / Q000096:biosynthesis*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D013379:Substrate Specificity; D013394:Sucrase-Isomaltase Complex / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D013394:Sucrase-Isomaltase Complex; D010450:Endopeptidases