Giorgio Semenza

A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems.

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.

The sucrase-isomaltase complex: Primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein

The complete primary structure (1827 amino acids) of rabbit intestinal pro-sucrase-isomaltase (pro-SI) was deduced from the sequence of a nearly full-length cDNA. Pro-SI is anchored in the membrane by a single 20 amino acid segment spanning the bilayer only once. The amino-terminal, cytoplasmic domain consists of 12 amino acids and is not preceded by a cleaved leader sequence. This suggests a dual role for the membrane-spanning segment as an uncleaved signal for membrane insertion. This is followed by a 22 residue serine/threonine-rich, probably glycosylated, stretch, presumably forming the stalk on which the globular, catalytic domains are directed into the intestinal lumen. Following this is a high degree of homology between the isomaltase and sucrase portions (41% amino acid identity), indicating that pro-SI evolved by partial gene duplication.

Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy

We have worked out a procedure for covalent binding of native biomacromolecules on flat gold surfaces for scanning probe microscopy in aqueous buffer solutions and for other nanotechnological applications, such as the direct measurement of interaction forces between immobilized macromolecules, of their elastomechanical properties, etc. It is based on the covalent immobilization of amino group-containing biomolecules (e.g., proteins, phospholipids) onto atomically flat gold surfaces via omega-functionalized self-assembled monolayers. We present the synthesis of the parent compound, dithio-bis(succinimidylundecanoate) (DSU), and a detailed study of the chemical and physical properties of the monolayer it forms spontaneously on Au(111). Scanning tunneling microscopy and atomic force microscopy (AFM) revealed a monolayer arrangement with the well-known depressions that are known to stem from an etch process during the self-assembly. The total density of the omega-N-hydroxysuccinimidyl groups on atomically flat gold was 585 pmol/cm(2), as determined by chemisorption of (14)C-labeled DSU. This corresponded to approximately 75% of the maximum density of the omega-unsubstituted alkanethiol. Measurements of the kinetics of monolayer formation showed a very fast initial phase, with total coverage within 30 S. A subsequent slower rearrangement of the chemisorbed molecules, as indicated by AFM, led to a decrease in the number of monolayer depressions in approximately 60 min. The rate of hydrolysis of the omega-N-hydroxysuccinimide groups at the monolayer/water interface was found to be very slow, even at moderately alkaline pH values. Furthermore, the binding of low-molecular-weight amines and of a model protein was investigated in detail.

ACQUIRED MILK INTOLERANCE IN THE ADULT CAUSED BY LACTOSE MALABSORPTION DUE TO A SELECTIVE DEFICIENCY OF INTESTINAL LACTASE ACTIVITY.

C HRONIC diarrhea due to deficient absorption of certain disaccharides has been described recently in infants and children as new inborn errors of metabolism [ 7-71. These syndromes have been variously termed disaccharide malabsorption, hereditary disaccharide intolerance and deficiency of sugar splitting intestinal enzymes. If unrecognized, the course may be steadily downhill, terminating in death of the infant; however, if the noxious sugar is eliminated from the diet, the disorders are essentially benign. Congenital lactose intolerance due to lactose malabsorption, becoming asymptomatic following the institution of a lactose-free (i.e., milkfree) diet, was first described in two siblings by Holzel et al. in 1959 [8] and named “alactasia” [8-701. Since then fourteen such cases have been recorded in the literature [6,8-731. Deficient intestinal lactase activity has been demonstrated recently [ 731. Congenital lactose intolerance associated with lactosuria is probably a distinct and more complex syndrome based on a different pathogenetic mechanism. It was first described by Durand in ‘1958 [ 741, a year before Holzel’s report appeared, as consisting of diarrhea, lactosuria, occasional aminoaciduria, renal acidosis and questionably related autopsy findings in the kidney, liver and central nervous system. Fifteen such patients have been described in the literature [9,74-201, but in none have intestinal enzyme studies been carried out. Twelve of the fifteen infants died despite the institution of a lactose-free diet. Lactose tolerance tests were performed in four infants [9,74,75,78], and the results showed a normal increase in blood reducing substances. This increase may be due to increased absorption of intact lactose, which in turn has a toxic effect on the whole organism, especially the kidneys. At present it appears best to separate the puzzling syndrome described by Durand from that of “benign” lactose intolerance described by Holzel and to regard Durand’s syndrome as a seperate entity rather than a more severe form of Holzel’s entity. Congenital sucrose intolerance due to sucrose malabsorption was first reported in 1960 by Weijers et al. in three children, in one of whom maltose tolerance was also poor [27,77]. Five patients with sucrose intolerance described by Prader et al. in 1961 [22] were later shown to have an additional intolerance to isomaltose and palatinose [23-251 which are split by the same enzyme [26]. A total of thirty-four cases of congenital sucrose intolerance have now been

The small-intestinal Na+,d-glucose cotransporter: An asymmetric gated channel (or pore) responsive to ΔΨ

SummaryAtΔψ≈0,d-glucose influx into, and efflux out of, membrane vesicles from small-intestinal brush borders are affected by trans Na+ and transd-glucose to different extents.d-glucose influx and efflux respond toΔψ (negative at the trans side) to different extents. The small-intestinal Na+,d-glucose cotransporter, is thus functionally asymmetric. This is not unexpected, in view of the structural asymmetry previously found. The characteristics of theΔψ of transinhibition byd-glucose are compatible with the mobile part of the cotransporter bearing a negative charge of at least 1 (in the substrate-free form). They are not compatible with its mobile part being electrically neutral. Pertinent equations are given in the Appendix. Partial Clelands kinetic analysis and other criteria rule out (Iso) Ping Pong mechanisms, and makes likely a Preferred Ordered mechanism, with Naout+ binding to the cotransporter prior to the sugarout. A likely model is proposed aimed at providing a mechanism of flux coupling and active accumulation.

Similarity in effects of Na+ gradients and membrane potentials ond-glucose transport by, and phlorizin binding to, vesicles derived from brush borders of rabbit intestinal mucosal cells

SummaryBoth the presence of sodium and of an electrical potential difference across the membrane have been found to be necessary in order to achieve optimald-glucose-protectable phlorizin binding to brush border membranes from rabbit small intestine. The effect of

A hydrophobic form of the small-intestinal sucrase-isomaltase complex

\Delta \tilde \mu _{Na}

Reconstitution of a sucrase‐mediated sugar transport system in lipid membranes

on phlorizin binding shows a close similarity to that ond-glucose transport, confirming that phlorizin is indeed bound to thed-glucose transporting protein. Possible modulations of binding by a transmembrane potential are discussed on the basis of some models.