A Simplified Procedure for the Isolation of the Sucrase · Isomaltase Complex from Rabbit Intestine Its Amino‐Acid and Sugar Composition

Abstract

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G-200 chromatography, has a molecular weight of approximately 220000 [2,3,8] (and Mosimann, H. and Semenza, G., unpublished results).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...

Dissociation of Small‐Intestinal Sucrase. Isomaltase Complex into Enzymatically Active Subunits

“…isomaltase complex was prepared from rabbit small intestine as described [5,11] and sucrase and isomaltase activities were determined with the Tris -glucose-oxidase -peroxidase reagent [I 21 with sucrose and palatinose as respective substrates. The isomaltase activity will be expressed as palatinose units throughout.…”

“…The isomaltase activity will be expressed as palatinose units throughout. Protein was measured according to Lowry et al [13], or from absorbance at 279 nm [11,14]. Free amino groups were determined with 2,4,6-trinitrobenzenesulfonic acid as described by Habeeb [I 51.…”

Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

“…Two of the dissacharidases, sucrase (EC 3.2.1.48) and isomaltase (EC 3.2.1.10), have been intensively studied. These enzymes represent distinct subunits in a dimeric protein complex which has been isolated from rabbit (Cogoli et al, 1972) and from human intestine (Conklin et al, 1975). Both subunits are glycoproteins (Cogoli et al, 1973) and have almost equal molecular weights of between 110000 and 140000 (Cogoli et al, 1973;Mosimann et al, 1973;Conklin et al, 1975).…”

Cell‐free synthesis of the one‐chain precursor of a major intrinsic protein complex of the small‐intestinal brush border membrane (pro‐sucrase—isomaltase)

“…At the moment it cannot be decided whether this indicates the processing of a (still hypothetical) pre-pro-S1 or whether the higher-mobility band arises from the other one by a proteolysis unrelated to the processes of translation and membrane insertion. Interpretation of this mobility change is complicated, at this stage, by the possibility that these polypeptides might be glycosylated, the sugar moieties of rabbit SI accounting for -15% of its weight [24,25].…”