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 amino 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) (15O/,) 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.The sucrase isomaltase complex is associated with, or a building block of, the membrane of smallintestinal brush borders (for a review see [l]). It is one of the relatively few membrane proteins which can be solubilized and isolated in good yield [2,3]. The major reason for our interest in this oligo-enzyme complex is its close relationship with the transport of some sugars across the brush border membrane [4-6] and the demonstration of its roIe as specific sugar carrier on artificial lipid membranes [7].Rabbit small intestinal sucrase isomaltase complex, as obtained by papain solubilization and Sephadex G-200 chromatography, has a molecular weight of approximately 220000 [2,3,8] (and Mosimann, H. and Semenza, G., unpublished results). As a further step towards its chemical characterisation we report here a simplified isolation procedure and data on its chemical composition.
EXPERIMENTAL PROCEDURE
Amino-Acid CompositionPrior to hydrolysis the protein samples were dialyzed against water. Their concentrations were and as confirmed by us, referring to the weight of protein samples which had been exhaustively dialyzed and dried under vacuum a t 80 "C in a "drying pistol"). Duplicate analyses were made with hydrolysis times of 24, 48, 72, 96 h, respectively. The protein samples (500 pg) were hydrolyzed in 2 ml 6 N HCl under vacuum a t 110 "C, norleucine being used as internal standard. The amino acids were determined on a BC-200 Analyzer from Calbiochem.Tyrosine and tryptophan were determined by the methods of Bencze and Schmid [lo] Total half-cystine and cysteine was determined by four different methods. Firstly, with 5,5'-dithiobis-(2-nitrobezoic acid) according to Ellmann [la] after denaturation and reduction of the protein in 8 M urea with 2-mercaptoethanol (65 nmol/mg protein at 37 "C for 1. h under nitrogen). Secondly it was determined as aminoethyl-cysteine [15] ; in this method several experiments were made using different concentrations of guanidine hydrochhride or urea as denaturating agent and mercaptoethanol or dithiothreitol as reducing agent. The highest yield was ob...
