Postinsertional processing of sucrase-alpha-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin.
Sucrase-alpha-dextrinase, a hybrid digestive carbohydrase of the intestinal brush border, is initially synthesized and transported to the surface membrane as a single-chain glycoprotein, P, which is then cleaved to alpha- and beta-subunits, presumably by one or more pancreatic proteases. However, efforts to convert P under controlled conditions to authentic alpha and beta have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presence of biliary-pancreatic secretions revealed only the 230-kDa P precursor. Restoration of intestinal flow converted the brush border P to the alpha- (140 kDa) and beta- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeled in vivo, were exposed to a mixture of pancreatic proteases at physiological concentrations, P was converted to authentic alpha and beta, but only trypsin was responsible for the conversion. Kinetic analysis in prelabeled isolated brush-border vesicles revealed the appearance of several intermediate species (205-145 kDa) produced either by endogenous membrane proteases or by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursors to the final authentic alpha- and beta-subunits. Based on the alpha- to beta radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the beta-subunit (alpha-to-beta molar ratio = 0.77). The conversion of P to the alpha- and beta-units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunits involving a complex series of cleavage steps.
Shapiro
G L
GL
Department of Medicine, Stanford University School of Medicine, California 94305.
Bulow
S D
SD
Conklin
K A
KA
Scheving
L A
LA
Gray
G M
GM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D001345:Autoradiography; D001659:Biliary Tract / Q000378:metabolism; D003720:Densitometry; D004386:Duodenum / Q000201:enzymology; D004792:Enzyme Precursors / Q000235:genetics*; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D010196:Pancreatic Elastase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D012995:Solubility; D013394:Sucrase-Isomaltase Complex / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism / Q000502:physiology*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004792:Enzyme Precursors; D008562:Membrane Glycoproteins; D013394:Sucrase-Isomaltase Complex; D010196:Pancreatic Elastase; D014357:Trypsin