G. Semenza

Studies on intestinal sucrace and on intestinal sugar transport. V. Isolaiton and properties of sucrase-isomaltase from rabbit small intestine☆☆☆

Abstract 1. 1.|The sucrase isomaltase complex has been isolated from rabbit small intestine. The procedure involves urea extraction of the whole intact small intestine, papain solubilization, and chromatography on Sephadex G-200 and polyacrylamide-gel (Bio-Gel P-300). 2. 2.|The chromatography on Sephadex G-200 is apparently based on an interaction of the substrate-enzyme type. 3. 3.|Sucrase-isomaltase, as isolated here, carries at least two substrate sites: one splitting sucrose and maltose, the other splitting isomaltose, palatinose and probably maltose also. 4. 4.|Na + is a non-essential activator. It increases the maximum velocity by some 20–30%, with minor or no change of the apparent K m for sucrose. 5. 5.|Tris is a strong fully competitive inhibitor. 6. 6.|The inhibition by some metals, and the lack of inhibition by other metals or by p -chloromercuribenzoate, indicate that SH groups are not required for full enzyme activity, whereas an imidazole group may be required.

The preparation of lactase and glucoamylase of rat small intestine

Abstract 1. 1.|Both lactase and glucoamylase were obtained in a highly purified form from the small intestine of baby rats, by papain solubilisation followed by chromatography on Sepharose, Sephadex and DEAE-cellulose. Some properties of these enzymes were investigated. 2. 2.|Attempts at affecting the levels of small intestinal lactase activity to the diet of baby rats failed. 3. 3.|The multiplicity of human intestinal lactases was also briefly investigated (see appendix ).

MULTIPLICITY OF HUMAN INTESTINAL DISACCHARIDASES. I. CHROMATOGRAPHIC SEPARATION OF MALTASES AND OF TWO LACTASES.

Abstract 1. 1. Disaccharidases from normal human intestinal mucosa have been solubilised by “autolysis” and/or papain (EC 3.4.4.10) digestion and have been partially separated by gel filtration on Sephadex G-200. 2. 2. With both types of solubilization used several maltases (maltose glucohydrolases) were found, some of which also have sucrase (sucrose glucohydrolase) of isomaltase (isomaltose glucohydrolase) and palatinase activities. 3. 3. With the same technique the existence of two lactases (lactose galactohydrolases) was demonstrated. Both of them attack cellobiose also. No other cellobiase (cellobiose glucosehydrolase) was found.

MULTIPLICITY OF HUMAN INTESTINAL DISACCHARIDASES. II. CHARACTERIZATION OF THE INDIVIDUAL MALTASES.

Abstract 1. 1. Human intestinal maltases (maltose glucohydrolases) obtained by solubilization and Sephadex chromatography were examined. Their heat stabilities, substrate specificities, pH-activity curves and Na + activation, as well as their chromatographic behaviour, indicate that 5 maltases are present in normal human intestine. Of these, two hydrolyse sucrose as well. One splits maltose, isomaltose and palatinose. 2. 2. The properties investigated did not apparently change during the solubilization step. 3. 3. The multiplicity of these maltases does not seem to arise during solubilization.

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

The dimeric sucrase-isomaltase complex (SI) (M,, 275 000 [l];M, of the subunits by SDS-PAGE, 120 000 and 140 000, respectively [2]) accounts for -10% of the intrinsic proteins of the brush border membrane of the small-intestine. The bulk of the protein mass is exposed to the luminal side of the membrane [2]. Its positioning has been elucidated as follows: (i) The isomaltase (I) subunit is anchored to the membrane via a highly hydrophobic loop located near the N-terminus of the polypeptide chain [2,3]; the segment between residues -12 and -60, which includes a Pro at position 35, is nearly totally in an o-helical configuration and presumably crosses the membrane bilayer twice [4]. (ii) No direct interaction of the sucrase (S) subunit with the membrane fabric could be detected [2]. (iii) The Nand C-termini of sucrase and the C-terminus of isomaltase are exposed to the luminal side [2]. (iv) The segment 1-l 1 of isomaltase is also located at the luminal side (Thr-1 1 is glycosylated [3,5] and the N-terminus can be labeled by an impermeant reagent [6]). particular arrangement of the SI complex, the homology between the 2 subunits (review [7,8]) and their related or common hormonal control (review [7]), one of us suggested in 1978 the ‘one-chain, twoactive-sites precursor hypothesis’ [9]. According to it, the 2 subunits have arisen from a common ancestor gene (coding for an isomaltase with maltase activity) by partial gene duplication giving rise to one polypeptide chain carrying two identical active sites; subsequent mutation(s) changed the substrate specificity of one of the sites: while maltose would still be accepted and hydrolysed, isomaltose would not be so any more, whereas sucrose would now be hydrolysed. This ‘one-chain, two active-sites sucrase-isomaltase’ (pro-SI) would be inserted into the membrane during synthesis and then split into the two chains of the ‘final’ SI complex by extracellular (e.g., pancreatic) proteases.

Studies on intestinal sucrase and sugar transport. VII. A method for measuring intestinal uptake the absorption of the anomeric forms of some monosaccharides

Abstract 1. 1. A procedure for measuring intestinal uptake is presented. It allows randomization of small-intestinal pieces, yields relatively small ‘extracellular space’ and allows the determination of unidirectional flux. 2. 2. The intestinal uptake of the anomeric forms of some monosaccharides has been measured. The β-form of 6-deoxy- d -glucose is better adsorbed than its α-form. In the case of d -glucose and of 3-O- methyl- d -glucose , no statistically significant difference could be ascertained between the anomeric forms. 3. 3. Some speculations on the possible role of sucrase and of mutarotase during the intestinal absorption of sugars are presented.

Studies on intestinal sucrase and intestinal sugar transport VI. Liberation of α-glucose by sucrase and isomaltase from the glycone moeity of the substrates☆

Abstract 1. 1.|Sucrase isomaltase isolated from rabbit intestine does not have detectable mutarotase activity. 2. 2.|The glucose which is liberated by either sucrase or isomaltase from the glycone moiety of the substrates retains the α-configuration of C-1. 3. 3.|The procedure developed to obtain these results involves freeze-drying of the samples, silylation and gas chromatographic separation.

Immunochemical study of isolated human and rabbit intestinal sucrase

Abstract 1. 1. Sucrase-isomaltase was isolated from human and from rabbit small intestinal mucosa. 2. 2. Antisera to these preparations were produced. 3. 3. By immunodiffusion and tube precipitation techniques there was shown to be immunological cross reactivity between these functionally related enzyme complexes. 4. 4. No inhibition of sucrase activity was detected upon preincubation with either specific or cross reacting antiserum.

ANCHORING AND BIOSYNTHESIS OF THE MAJOR INTRINSIC PROTEIN OF THE SMALL-INTESTINAL BRUSH BORDER MEMBRANE

Publisher Summary The sucrase-isomaltase complex (SI) is the major intrinsic protein of the small-intestinal brush border membrane, accounting for approximately 10% of the total protein. Any mechanism of biosynthesis and membrane insertion of SI must explain its positioning, in particular the peripheral location of S and the mode of anchoring of I. In addition, it should accommodate the analogy—indeed partial homology—of the two subunits and their common or related biological control mechanism. The existence of pro-SI as the immediate, fully enzymatically active precursor of final SI is well established. Pulse chase experiments show a high molecular weight band appearing in the Golgi membranes first and then in the brush borders; elastase treatment splits it into bands of mobilities close to those of the final subunits S and I. The main events in the biosynthesis, insertion, glycosylation and processing of pro-SI→SI seem, therefore, to be well established. They provide a logical explanation of the positioning of final SI and also of the possible genetic mechanisms underlying human sucrose–isomaltose malabsorption.