Summary
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°C using a dynamic approach which involves linearregression 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°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv 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 ofd-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.
Similar content being viewed by others
References
Alvarado, F., Brot-Laroche, E., Delhomme, B., Serrano, M.A., Supplisson, S. 1986. Heterogeneity of sodium-activated-d-glucose transport systems in the intestinal brush border membrane: Physiological implications.Boll. Soc. Ital. Biol. Exp. LXII:110–132
Berteloot, A. 1984. Characteristics of glutamic acid transport by rabbit intestinal brush border membrane vesicles. Effects of Na+-, K+- and H+-gradients.Biochim. Biophys. Acta 775:129–140
Berteloot, A. 1986. Highly permeant anions and glucose uptake as an alternative for quantitative generation and estimation of membrane potential differences in brush border membrane vesicles.Biochim. Biophys. Acta 857:180–188
Berteloot, A. 1986. Membrane potential dependency of glutamic acid transport in rabbit jejunal brush border membrane vesicles. K+ and H+ effects.Biochim. Biophys. Acta 861:447–456
Berteloot, A., Malo, C., Breton, S., Brunette, M. 1991. A fast sampling, rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose, transport in human jejunal brush-border membrane vesicles.J. Membrane Biol. 122:111–125
Berteloot, A., Semenza, G. 1990. Advantages and limitations of vesicles for the characterization and the kinetic analysis of transport systems.Methods Enzymol. 192:409–437
Brot-Laroche, E., Serrano, M.A., Delhomme, B., Alvarado, F. 1986. Temperature sensitivity and substrate specificity of two distinct Na+-activatedd-glucose transport systems in guinea pig jejunal brush border memrane vesicles.J. Biol. Chem. 261:6168–6176
Dorando, F.C., Crane, R.K. 1984. Studies of the kinetics of Na+ gradient-coupled glucose transport as found in brush-border membrane vesicles from rabbit jejunum.Biochim. Biophys. Acta 772:273–287
Dowd, J.E., Riggs, D.S. 1965. A comparison of estimates of Michaelis-Menten kinetic constants from various linear transformations.J. Biol. Chem. 240:863–869
Freeman, H.J., Quamme, G.A. 1986. Age-related changes in sodium-dependent glucose transport in rat small intestine.Am J. Physiol. 251:G208-G217
Garfinkel, D., Fegley, K.A. 1984. Fitting physiological models to data.Am. J. Physiol. 246:R641-R650
Garfinkel, L., Kohn, M.C., Garfinkel, D. 1977. Systems analysis in enzyme kinetics.Crit. Rev. Bioeng. 2:329–361
Harig, J.M., Barry, J.A., Rajendran, V.M., Soergel, K.H., Ramaswamy, K. 1989.d-glucose andl-leucine transport by human intestinal brush-border membrane vesicles.Am. J. Physiol. 256:G618-G623
Hopfer, U. 1977. Kinetics of Na+-dependentd-glucose transport.J. Supramol. Struct. 7:1–13
Hopfer, U. 1981. Kinetic criteria for carrier-mediated transport mechanisms in membrane vesicles.Fed. Proc. 40:2480–2485
Hopfer, U. 1981. Kinetic features of cotransport mechanisms under isotope exchange conditions.Membr. Biochem. 4:11–29
Hopfer, U., Nelson, K., Perrotto, J., Isselbacher, K.J. 1973. Glucose transport in isolated brush border membrane from rat small intestine.J. Biol. Chem. 248:25–32
Kaunitz, J.D., Wright, E.M. 1984. Kinetics of sodiumd-glucose cotransport in bovine intestinal brush border vesicles.J. Membrane Biol. 79:41–51
Kessler, M., Tannenbaum, V., Tannenbaum, C. 1978. A simple apparatus for performing short-time (1–2 seconds) uptake measurements in small volumes; its application tod-glucose transport studies in brush border vesicles from rabbit jejunum and ileum.Biochim. Biophys. Acta 509:348–359
Kimmich, G.A. 1990. Membrane potentials and the mechanism of intestinal Na+-dependent sugar transport.J. Membrane. Biol. 114:1–27
Kunst, A., Draeger, B., Ziegenhorn, J. 1984. UV-methods with hexokinase and glucose-6-phosphate deshydrogenase.In: Methods of Enzymatic Analysis (3rd ED.). Vol. 6. pp. 163–172. Metabolites I: Carbohydrates. H. U.Bergmeyer, editor. Verlag Chemie, Weinheim
Leatherbarrow, R.J. 1990. Use of nonlinear regression to analyze enzyme kinetic data: Application to situations of substrate contamination and background subtraction.Anal. Biochem. 184:274–278
Lücke, H., Berner, W., Menge, H., Murer., H. 1978. Sugar transport by brush border membrane vesicles isolated from human small intestine.Pfluegers Arch 373:243–248
Maes, F.W. 1984. Problems with linear regression analysis of pharmacological and biochemical data.J. Theor. Biol. 111:817–819
Malo, C. 1988. Kinetic evidence for heterogeneity in Na+-d-glucose cotransport systems in the normal human fetal small intestine.Biochim. Biophys. Acta 938:181–188
Malo, C. 1990. Separation of two distinct Na+/d-glucose cotransport systems in the human fetal jejunum by means of their differential specificity for 3-O-methylglucose.Biochim. Biophys. Acta 1022:8–16
Malo, C., Berteloot, A. 1987. Proximo-distal gradient of Na+-dependentd-glucose transport activity in the brush border membrane vesicles from the human fetal small intestine.Febs Lett. 220:201–205
Mannervik, B. 1981. Design and analysis of kinetic experiments for discrimination between rivals models.In: Kinetic Data Analysis. Design and Analysis of Enzyme and Pharmacokinetic Expriments. L. Endrenyi, editor. pp. 235–270. Plenum, New York-London
Orsi, B.A., Tripton, K.F. 1979. Kinetic analysis of progress curves.Methods Enzymol. 63:159–183
Robinson, J.W.L., Van Melle, G.J. 1983. Kinetics of the sodium/B-methyl-d-glucoside contransport system in the guinea-pig small intestine.J. Physiol. 344:177–187
Sagnella, G.A. 1985. Model fitting, parameter estimation, linear and non-linear regression.Trends Biol. Sci. 10: 100–103
Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J., Crane, R.K. 1973. Purification of the human intestinal brush border membrane.Biochim. Biophys. Acta 323:98–112
Semenza, G., Kessler, M. Hosang, M., Weber, J., Schmidt, U. 1984. Biochemistry of the Na+-d-glucose cotransport of the small-intestinal brush-border membrane. The state of the art in 1984.Biochim. Biophys. Acta 779:343–379
Van Melle, G.J., Robinson, J.W.L. 1981. A systematic approach to the analysis of intestinal transport kinetics.J. Physiol. (Paris) 77:1011–1016
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Malo, C., Berteloot, A. 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. J. Membrain Biol. 122, 127–141 (1991). https://doi.org/10.1007/BF01872636
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF01872636