The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters

Abstract

A new plot is described for analysing the results of kinetic experiments in which the Michaelis-Menten equation is obeyed. Observations are plotted as lines in parameter space, instead of points in observation space. With appropriate modifications the plot is applicable to most problems of interest to the enzyme kineticist. It has the following advantages over traditional methods of plotting kinetic results: it is very simple to construct, because it is composed entirely of straight lines and requires no calculation or mathematical tables; the kinetic constants are read off the plot directly, again without calculation; it may be used during the course of an experiment to judge the success of the experiment, and to modify the experimental design; it provides clear and accurate information about the quality of the observations, and identifies aberrant observations; it provides a clear indication of the precision of the kinetic constants; constructed with care, it provides unbiased estimates of the kinetic constants, the same as those provided by a computer program; it may be used to simulate results for illustrative purposes very rapidly and simply.

A novel doxorubicin-glucuronide prodrug DOX-GA3 for tumour-selective chemotherapy: distribution and efficacy in experimental human ovarian cancer

“…After incubation for 30 min at 37˚C the reaction was stopped by adding methanol. Samples were analysed by HPLC and K m and V max values were calculated from direct linear plots as described by Eisenthal and Cornish-Bowden (Eisenthal and Cornish-Bowden, 1974).…”

“…The prodrug, incubated at the clinically relevant concentrations of 100 µM for DOX-GA3 and of 1 µg ml -1 for the enzyme, was hydrolysed at pH 6.8 and 37˚C with a t 1 / 2 of 140 min. Based on the in vitro cleavage experiments at different prodrug concentrations at pH 6.8 and at 37˚C, V max and K m were calculated from direct linear plots as described by Eisenthal (Eisenthal and Cornish-Bowden, 1974). For DOX-GA3 a V max of 25.0 µmol min -1 mg -1 and a K m of 1100 µM was found.…”

A role of Lys614 in the sulfotransferase activity of human heparan sulfate N‐deacetylase/N‐sulfotransferase

“…The radioactivities of the eluates were determined by liquid scintillation. KIn.PAPS was determined by the direct linear plot [14]. The enzyme concentrations were determined using a molar absorbance coefficient (e2~ = 60 790) calculated from the content of tryptophan and tyrosine in the enzyme.…”

Adenylate cyclase activity in permeabilised yeast

“…AC was assayed at 30°C by isolating [5] [7] from 5-7 datum points between 0.15 and 3.0 mM ATP. The ranges of the middle thirds of the sets of estimates [7] for V m and Vm/K m are shown by error bars in fig.2.…”

“…The ranges of the middle thirds of the sets of estimates [7] for V m and Vm/K m are shown by error bars in fig.2. K m was calculated as the ratio of V m and Vm/K m. Reagents not described in [5] were of analytical grade.…”