Abstract
D-Glucose and D-galactose influx and efflux rates in human erythrocytes were studied using infinite-cis and zero-trans assay methods. It was found that insulin decreased the infinite-cis Km for both D-glucose and D-galactose influx by 44 and 56%, respectively, while the Vmax was unchanged. The Km for D-glucose efflux in the presence of insulin decreased by 47% when compared to controls, and the change in Vmax was statistically insignificant. If insulin receptors were first down regulated, and then influx and efflux assays were performed, decreases in the infinite-cis and zero-trans Km values were also observed in the absence of exogenous insulin. These affinity changes were not due to persistent surface insulin receptor occupation by the insulin which was used to induce down-regulation. These affinity changes were comparable to those observed in non-down-regulated cells in the presence of insulin.
Highlights
Using modificationsof the zero-trans and infinite-cistransport assays, we have examined the effects of insulin on D
D-Glucose and D-galactoseinflux and efflux rates in human erythrocyteswere studied usinginfinite-cisand zero-tram assay methods.It was foundthatinsulin decreased theinfinite-cis K, for both D-glucose anDdgalactose influx by 44 and 56%, respectively, while uptake and zero-trans efflux without significantly changing the V
Weiser et al [19] have described an integrated rate equation which can be used to analyze the uptake time course by transforming it into a linear plot. Information from this plot canbe used to determine the initial velocity of glucose influx andthe internal Dglucose concentration a t which the netuptake rate is reduced to onehalf of the initial velocity
Summary
Compared to controls, and the change in V, was statistically insignificant. If insulin receptors were first down regulated, and thiennflux and efflux assayswere performed, decreases in thine finite-cis and zero-tram. Information from this plot canbe used to determine the initial velocity of glucose influx (zero-trans) andthe internal Dglucose concentration a t which the netuptake rate is reduced to onehalf of the initial velocity The latter value is related to the affinity of the inner binding site of the membrane for glucose and is known as the infinite-cis K, [18]. These methods both require the rapid cessation of transport at specific times in order to accurately determine time courses In both infinite-cis and zero-trans assays, suspensions of cells were transferred into a large volume of 0 “C stopping solution containing 1 p~ mercuric chloride, 1.25 mM potassium iodide, and 0.1 mM phloretin [3,4,5]. Standard errors determined by this method were used for significance testing using the t-distribution with the appropriate degrees of freedom
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