Hans Wacker

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.

Incorporation of hydrophobic aminopeptidase from hog kidney into egg lecithin liposomes: Number and orientation of aminopeptidase molecules in the lecithin vesicles

The brush border of renal tubuli contain an aminopeptidase, which is bound to, or is a building block of, the apical membrane [2,3]. Extraction with Triton X-100 yields an aggregated form of the enzyme which can be transformed into a low molecular weight form by the action of trypsin [4]. During this transformation a fragment of approximately 10 000 daltons is released; this probably represents the hydrophobic portion which anchors the protein to the lipid matrix of the original membrane [4]. This ‘anchor’ is likely to be of importance in reconstitution experiments, as has been shown for other amphipatic proteins [S] . Recently, a membrane-bound aminopeptidase’from the small intestine has been shown to have a hydrophobic region [6]. Peptides cross the brush border membrane (of the small intestine) through route(s) which are different from those utilised by qmino acids (for a review, see [7] ). Utilising pure membrane vesicles from intestinal brush border membranes [8], it has been shown in this laboratory that peptides may cross the brush border at the same time as they are hydrolysed. The interest in studying the mode of incorporation of aminopeptidase into monolamellar vesicles of pure lipids is, therefore, obvious. To this goal we utilised aminopeptidase from (hog) kidney, rather than intestine, because the former can be obtained more easily in a fully undegraded form; this is due to the lack of very powerful proteases in kidney brush

[31] Sucrase-isomaltase of the small-intestinal brush border membrane: Assembly and biosynthesis

Publisher Summary This chapter describes the application of chemical labeling reagents developed to gain a better understanding of sucrase-isomaltase enzyme complex, and how this anchoring peptide is structured and folded within the membrane. Knowledge of the folding pattern is an important step toward formulating a possible mechanism of biosynthesis and membrane insertion of this enzyme complex. The chapter also discusses the conditions for the cell-free synthesis of the precursor TM and a possible explanation for the particular topological arrangement of sucrase-isomaltase. This technique, therefore, represents a valuable complement to surface-labeling techniques of membranes. The chapter suggests that pro-sucrase-isomaltase (pro-SI) is synthesized with a leader peptide–an assumption justified by the fact that the N-terminal of I is located on the extracellular side. A first hairpin would establish the first insertion into the membrane, and a second hydrophobic hydrophilic hairpin would follow immediately, and the rest of pro-sucrase-isomaltase could be synthesized and extruded either completely or up to another hydrophobic segment situated closely to the C terminus.

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.