The intestinal transport of sugars and amino acids seems to follow Michaelis-Menten kinetics, but the presence of unstirred water layers at the outer face of the brush border membrane may distort kinetic measurements. According to current theory, the capacity parameter, Jmc max would not be affected, but the Kt would be increased to a higher value, Kt ', in proportion to the thickness of the unstirred water layer, d. We reasoned that by increasing the shaking rate in the tissue accumulation method, d might drop to such small values that Kt ' would fall to a constant level practically equal to the "true" Kt. We measured D-galactose influx into rings of everted hamster intestine as a function of both the substrate concentration and the shaking rate. Our results show that as the circular stirring rate increases from 0.38--6.2 Hz, J mc max remains constant, as expected, but Kt ' first drops, then levels off to reach a plateau between 2 and 6.2 Hz. We conclude that the average Kt values in this frequency range (Kt = 7.4 mM) represent the true transport Kt. Furthermore, all previous kinetic work performed in our laboratory has been carried out under identical conditions, including shaking rates of 4 Hz. The validity of our preceding results is thus upheld.

SUMMARY1. Hydrolase-related transport was re-investigated in hamster small intestine by the tissue accumulation method.2. The Na+-dependent, phlorizin-sensitive monosaccharide transport system saturates with 30 mM-D-glucose. According to the hydrolase-related transport hypothesis, additional glucose units will be taken up if they are given in the form of a disaccharide susceptible to hydrolysis. But in experiments with [14C]sucrose we found no evidence for any such surplus glucose uptake.3. The uptake of '4C label from sucrose was abolished by using Tris, a strong inhibitor of sucrase, by adding competitive inhibitors of the D-glucose transport system (D-glucose, ,-methyl-D-glucopyranoside or phlorizin), and by substituting Li+ for the Na+ in the incubation medium.4. Glucose and fructose derived from sucrose did not enter the tissues in equimolar amounts: the glucose moiety was taken up much faster.5. We conclude that in hamster intestine there is no evidence for the existence of hydrolase-related transport with sucrose as the monosaccharide donor. The enzymatic hydrolysis of sucrose and the transport of its products, glucose and fructose, are two distinct events, acting sequentially.

Harmaline inhibition of sodium uptake and of sodium-dependent D-glucose transport was investigated using brush-border membrane vesicles from frozen rabbit jejunum. Under sodium-gradient conditions, "initial" D-glucose uptake (20 s) was inhibited by harmaline at concentrations above 0.5 mM, but at lower harmaline concentrations D-glucose uptake was stimulated by 10--15%. When a similar potassium gradient was used, harmaline had no effect. At concentrations up to 2 mM, harmaline did not alter the equilibrium uptake of D-glucose or D-mannitol. After pre-equilibration with sodium (25 mM), G-glucose uptake was inhibited at harmaline concentrations ranging from 0.1 to 2 mM. Sodium (10 mM) uptake was also inhibited by harmaline. Increasing the sodium concentration reduced the inhibitory effect of harmaline on tracer sodium uptake as well as on sodium-dependent D-glucose uptake. Similar to phlorizin, harmaline (1 mM) was able to prevent glucose-induced sodium influx across the brush-border membrane. Sodium uptake into brush-border membrane vesicles seems to be inhibited at lower harmaline concentrations than sodium-dependent D-glucose uptake. At high (2 mM) inhibitor concentrations, however, sodium-dependent glucose uptake is more strongly inhibited than sodium uptake. These results suggest that harmaline inhibits both sodium and sodium-dependent transport across intestinal brush-border membranes by interacting with specific sodium-binding sites.

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