Search

1. Several selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation onl5, in the reaction of carboxyl groups with carbodiimide ( + glycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem. 251, 3250–3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed. [ABSTRACT FROM AUTHOR]

Limited tryptic digestion of native sucrase. isomaltase complex produced a more rapid destruction of isomaltase activity than sucrase activity. It was possible to isolate a partially fragmented sucrase subunit in high yields with a specific activity twice that of the native complex. Amino acid and carbohydrate analyses are reported and compared with the results obtained for sucrase isomaltase complex and isomaltase subunit obtained by a different method. [ABSTRACT FROM AUTHOR]

1. The sucrase isomaltase complex from rabbit small intestine dissociated into its subunits upon reaction with citraconic anhydride. They can recombine after deacylation under mild acidic' conditions. 2. When citraconylated, the subunits could be separated and isolated in a catalytically active form. 3. The previously reported procedure for separation of the subunits by alkaline treatment at pH 9.6 is apparently not due to contaminating degradative enzymes (possibly still present at undetectable levels in the isolated sucrase isomaltase complex) but to the action of alkali. [ABSTRACT FROM AUTHOR]

1. Steady-state analysis of product inhibition shows that the most probable kinetic mechanism of intestinal sucrase is ping-pong bi-bi (ordered uni-bi for hydrolysis alone). 2. The site of activation by Na+ was tentatively localized at a level prior to the further trans- formation of the ‘glucose’ · enzyme complex. 3. Tris(hydroxymethyl)aminomethane competes with the substrate for the glucose subsite. 4. In the appendix a simple kinetic text is described for detecting or ruling out a mutual competition between inhibitors of different or of identical kinetic type. [ABSTRACT FROM AUTHOR]

A simplified procedure for the isolation of the sucrase · isomaltase complex from rabbit small intestine was worked out, which allows the preparation of this protein in a homogeneous form in good yield. The sucrase · isomaltase complex from rabbit small intestine is a glycoprotein. It is rich in acidic ammo acids; it has no free thiols in the native state but denaturation unmasks six thiols, The tyrosine/tryptophan ratio is 2. The sugar moiety(ies) (15%) is composed of D-glucose, D-galactose, D-mannose, fucose, glucosamine and galactosamine (the latter two are probably acetylated). The sucrase-isomaltase complex contains no neuraminic, uronic acid, lipids, phosphate, sulfate or zinc. The partial specific volume, calculated from the amino acid and sugar composition, agrees with that experimentally determined. The hydrophobicity coefficient of this membrane protein does not differ from that of other membrane bound or water soluble proteins. [ABSTRACT FROM AUTHOR]