Structural and functional correlates of sucrase-alpha-dextrinase in intact brush border membranes.
The structure and catalytic function of rat intestinal sucrase-alpha-dextrinase (sucrase-isomaltase) were characterized in intact brush border membranes by differential denaturation in 1% SDS at 4, 37, 45, 55, and 100 degrees C, analysis by acrylamide electrophoresis, and subsequent renaturation by transfer to nitrocellulose and in situ analyses of immunoactivity and catalytic activity (immunoblotting and catalytic blotting). Both the sucrase and alpha-dextrinase activities were associated with two mature oligomers, with sucrase predominantly in a 250-260-kDa unit and dextrinase in a 330-350-kDa unit. While sucrase activity declined progressively in response to increasing temperature to 45 degrees C due to loss of active sites, alpha-dextrinase activity increased reciprocally (Vmax +176%). Three principal monomeric products of postinsertional processing comprise the oligomers: alpha, 140 kDa, which carries the sucrase active site; beta, 125 kDa, harboring the dextrinase active site; and gamma, 110 kDa, produced by removal of 185 amino acid residues from the N-terminus of the alpha. Rather than being a simple hybrid dimer, membrane-associated sucrase-alpha-dextrinase appears to consist of two major oligomeric forms having complex structural associations that dramatically affect the availability of the active catalytic sites at the brush border membrane surface.
Department of Medicine, Beckman Center, Stanford University School of Medicine, California 94305.
J S
JS
Zhu
K A
KA
Conklin
L A
LA
Scheving
A J
AJ
Smith
G M
GM
Gray
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology; D008297:Male; D008871:Microvilli / Q000201:enzymology*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid; D013394:Sucrase-Isomaltase Complex / Q000235:genetics* / Q000378:metabolism; D013552:Swine
D006867:Hydrolases; D013394:Sucrase-Isomaltase Complex