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Limitations of the Eadie-Hofstee plot to estimate kinetic parameters of intestinal transport in the presence of an unstirred water layer

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Summary

In the presence of an intestinal unstirred water layer, the relationship between substrate concentration (C 1) and unidirectional flux (J d) is not described by the equation for a rectangular hyperbole. Accordingly, transformations of the Michaelis-Menten equation may not necessarily be linear and may lead to serious errors in the estimation of the affinity constant (K m) and maximal transport rate (J md ) of carrier-mediated processes. An equation has previously been derived which describedJ d under conditions of varying effective thickness or surface area of the unstirred water layer, the free diffusion coefficient of the probe molecule, and the distribution of transport sites along the villus. These theoretical curves have been analyzed by using the Eadie-Hofstee transformation (J d vs. Jd/C1) of the Michaelis-Menten equation. Use of this plot leads to serious discrepancies between the true and apparent affinity constants and between true and apparent maximal transport rates. These differences are further magnified by failure to correct for the contribution of passive permeation. The Eadie-Hofstee plot is of use, however, to infer certain qualitative characteristics of active transport processes, such as the variation in affinity constants and overlying resistance of the unstirred water layer at different sites along the villus and to predict the adequacy of the correction for the contribution of passive permeation.

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Abbreviations

C 1 :

Concentration of the probe molecule in the bulk phase

C 2 :

Concentration of the probe molecule at the aqueous-membrane interface

d :

Effective thickness of UWL

D :

Free diffusion coefficient

d n :

d atn th segment of the villus

f n :

Proportion of total carrier transport sites present on each segment of villus

J :

Unidirectional flux of probe molecule, uncorrected for surface area

J d :

Unidirectional flux of probe molecule determined experimentally, corrected for surface area

J md :

Maximal transport rate, corrected for surface area

J m*d :

Apparent maximal transport rate

J m :

Maximal transport rate, uncorrected for surface area

K m :

Michaelis constant (true affinity constant)

K *m :

Apparent affinity constant

K nm :

K m atnth segment of the villus

n :

The perpendicular height of the villus was divided into ten equal segments numberedn 1 ton 10

p :

Passive permeability coefficient

S m :

Functional surface area of the membrane

S w :

Effective surface of UWL

S nw :

S w atnth segment of the villus

UWL:

Intestinal unstirred water layer

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  1. Department of Medicine, University of Alberta, 9-112 Clinical Sciences Building, T6G 2G3, Edmonton, Alberta

    Alan B. R. Thomson

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  1. Alan B. R. Thomson
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Thomson, A.B.R. Limitations of the Eadie-Hofstee plot to estimate kinetic parameters of intestinal transport in the presence of an unstirred water layer. J. Membrain Biol. 47, 39–57 (1979). https://doi.org/10.1007/BF01869046

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  • DOI: https://doi.org/10.1007/BF01869046

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