Application of modified Lineweaver-Burk plots to studies of kinetics and regulation of radish 3-hydroxy-3-methylglutaryl-CoA reductase.

ABSTRACT

We propose the use of modified Lineweaver-Burk plots for the correct evaluation of Michaelis-Menten parameters in radioactive enzyme assays. A correction factor X for the translation of 1/S0 into 1/S is directly derived from the integrated Michaelis-Menten equation without the need of complicated calculations. In practice, this approach is favorably combined with an isotope dilution method which enhances the reliability of measurements at low substrate concentrations. The usefulness of the theoretical and practical approach is demonstrated in investigations of HMG-CoA reductase present in membrane fractions isolated from radish seedlings. The enzyme in the two main membrane fractions obtained by centrifugation at 16000 X g ( P16000 ) and at 105000 X g ( P105000 ) appears to be independently regulated by phytochrome and by phytohormones. Whereas active phytochrome decreases the apparent V of HMG-CoA reductase in the P105000 without affecting the Km, it increases the apparent Km in the P16000 . Kinetin treatment also results in a higher apparent Km of the enzyme in the P16000 fraction. Gibberellic acid and indoleacetic acid did not exhibit such a clear effect.