PROPERTIES AND KINETICS OF MEMBRANE-BOUND ENZYMES WHEN BOTH THE ENZYME AND SUBSTRATE ARE COMPONENTS OF THE SAME MICROSOMAL MEMBRANE - STUDIES OF LATHOSTEROL 5-DESATURASE

Microsomes isolated from rat liver contain an NADH-dependent lathoster ol B-desaturation system that catalyzes the introduction of a Delta(5) bond into lathosterol to form 7-dehydrocholesterol. Microsomes were p reloaded in vitro with liposomes composed of lathosterol and phosphati dylcholine in the presence of a high-speed supernatant (S-105) protein prior to enzyme assay. The desaturation led to a reaction that occurr ed in two distinct phases. That is, there was an initial burst of prod uct formation over an approximate time scale of 5 min that fell off, t hereafter to a steady state rate for over 30 min. The latter steady st ate phase was slower than the burst phase, because lateral diffusion o f the lathosterol substrate must occur before the next reaction can ta ke place. The total amount of the burst, which may be obtained by extr apolating the linear part of the curve in the steady state phase back to zero time, provides a means of obtaining the enzyme concentration i n terms of functional active sites. It was found that the kinetics bet ween enzyme and substrate within the same membrane also followed the u sual kinetic formalism of a Michaelis-Menten type reaction as in nonag gregated homogenous solution.