Analysis of kinetic data in transport studies: new insights from kinetic studies of Na(+)-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus.
Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991, J. Membrane Biol. 122:111-125), we have studied the kinetic characteristics of Na(+)-D-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35 degrees C using a dynamic approach which involves linear-regression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20 degrees C, in the presence of high substrate concentrations or 1 mM phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classical v versus S plot. All together, our results support the existence of a single Na(+)-D-glucose cotransport system in these membranes for which Na+ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989. Am J. Physiol. 256:8618-8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters of D-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved: (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.
Malo
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Department of Physiology, Faculty of Medicine, University of Montréal, Qué., Canada.
Berteloot
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D001692:Biological Transport / Q000502:physiology; D002458:Cell Fractionation; D005374:Filtration / Q000295:instrumentation / Q000379:methods*; D005947:Glucose / Q000493:pharmacokinetics*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D007583:Jejunum / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D008871:Microvilli / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D012964:Sodium / Q000493:pharmacokinetics*; D013696:Temperature
D012964:Sodium; D005947:Glucose