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

Abstract

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 a isomaltase complex) but to the action of alkali.The sucrase . isomaltase complex is a membranebound protein which translocates some sugars across natural [I, 21 and artificial [3] membranes. It is a glycoprotein composed of two similar, but not identical subunits of molecular weight (M,) approximately 112000 [5], one splitting sucrose and the other isomaltose as well as a number of other a-glucosides. It was possible to separate the two subunits by the action of denaturing agents [5], the denaturation being irreversible. A catalytically active subunit could be obtained only through degradation of the other subunit: isomaltase through mild alkaline treatment (which brought about an almost complete destruction of sucrase [4,6]) and sucrase through trypsin digestion (which destroyed almost completely isomaltase [7]).The purpose of this paper is to present a procedure allowing the separation of both subunits of the complex and their isolation iil a catalytically active form. The procedure is based on reaction of the protein amino groups with citraconic anhydride. This is known to lead to dissociation of other oligomeric proteins [X-101 through a drastic change of the electrostatic interaction among subunits. Citraconylation of sucrase isomaltase complex followed by separation and deacylation under mild acidic conditions leads to catalytically active subunits. out before separation of the subunits reformation of the complex occurs.We also present additional evidence that in the mild alkaline treatment which leads to the preparation of isomaltase (as reported previously [4,6]) no action of contaminating proteases is involved. MATERIALS AND METHODSThe sucrase . 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. 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. In this determination samples of control enzyme subjected to the same conditions as the citraconylated enzyme were assayed at the same time. All results are expressed with respect to such controls.Neutral carbohydrates were released from the isomaltase subunit by treatment with 2 N H2S04 in evacuated tubes at 100 "C for 4 h. The ...

Demonstration of sucrase-isomaltase complex in chick intestine.

“…Semenza et al (3,9,14) showed the properties of sucrase-isomaltase complex as follows: a sucrase-isomaltase complex is retained by Sephadex G-200 owing to a substrate-enzyme like interaction via its isomaltase site. The purified sucrase isomaltase complex had different catalytic sites for sucrase and isomaltase activities and sucrose and isomaltose were hydrolyzed at different sites; however , elec trophoresis or ultracentrifugation did not dissociate the complex into sucrase and isomaltase; as sucrase and isomaltase formed an enzyme-enzyme complex , if the deficiency of either sucrase or isomaltase was found, always both enzymes were deficient; moreover, they showed that sucrase-isomaltase complex was dissociated by citraconylation into sucrase and isomaltase without cleaving the covalent bond .…”

Tryptic Digestion of Native Small‐Intestinal Sucrase · Isomaltase Complex: Isolation of the Sucrase Subunit

“…isomaltase complex can also be separated through a drastic change of the surface charges by citraconylation. After decitraconylation they seem to recombine under appropriate conditions [9].…”

“…The two subunits can be separated by mild alkaline treatment [8] or by reaction with citraconic anhydride [9]. As a result of the alkaline treatment, a pure active isomaltase as well as an aggregate of inactive sucrase and a small amount of slightly active sucrase were isolated and characterized.…”

Ultrastructural basis of intestinal absorption.

“…These enzymes are known to constitute structural proteins of the membrane, their active sites protruding into the lumen from the membrane surface , being associated with polysaccharide chains (ODA and SEKI , 1966;NISHI and TAKESUE, 1978). Since it has been considered that sucrase and isomaltase form a complex in the microvillous membranes (BRAUN et al, 1975;NISHI and TAKESUE , 1978), and lactase and phloridin hydrolase also form a complex (COLOMBO et al, 1973), one possibility may be that these enzyme proteins assemble into intramembranous particles together with some other integral protein subunits to form a transport channel as mentioned before . If this interpretation is acceptable, it is conceivable that immediately after splitting from maltose by action of membrane maltase, for example, glucose could be transported across the membrane into the cytoplasm through the channel joined by the same maltase, thus implying the close coupling of absorption with the terminal digestion of sugar.…”