Markus Kessler

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 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.

A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes; its application to d-glucose transport studies in brush border vesicles from rabbit jejunum and ileum

An automated procedure allows uptake measurements with incubation times as short as 0.5 s and with volumes of 10--20 microliter. Using this technique the kinetic parameters Km and V of D-glucose transport in brush border vesicles from rabbit small intestine could be determined from unidirectional fluxes. A comparison of the data obtained from jejunum and from ileum shows that the Km for D-glucose is the same in both parts of the intestine, whereas the maximum flux is significantly larger in the jejunum.

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

Na+-dependent, potential-sensitive L-ascorbate transport across brush border membrane vesicles from kidney cortex.

\Delta \tilde \mu _{Na}

Nonelectrolyte Transport in Small Intestinal Membrane Vesicles. The Application of Filtration for Transport and Binding Studies

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.

THE Na+-GLUCOSE COTRANSPORTER OF THE SMALL INTESTINAL BRUSH BORDER MEMBRANE: AN ASYMMETRIC GATED CHANNEL (OR PORE) RESPONSIVE TO ΔΨ

In brush border vesicles from guinea pig small intestine L-ascorbate transport is Na+-dependent and electroneutral (in the presence of Na+, as shown by its lack of response to either positive or negative delta psi across the membrane). L-Ascorbate transporter has the kinetic characteristics of a mobile carrier (Km for L-ascorbate, 0.3 mM). D-Isoascorbate (erythorbate) seems to be another, but poorer, substrate of the same transporter. L-Ascorbate transport is subjected to heterologous inhibition by D-glucose.

Biochemistry of the Na+, d-glucose cotransporter of the small-intestinal brush-border membrane: The state of the art in 1984

l-Ascorbate is taken up into brush border vesicles from kidney cortex of rat, rabbit and guinea pig by an efficient, Na+-dependent and potential-sensitive transport process. This uptake shows saturation (Km:0.1–0.3 mM) and is strongly stimulated by low concentrations of N3−. Erythorbate (d-isoascorbate) seems to be another, but poorer, substrate of the same transporter.