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
Y. Takakuwa et al., 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, The Journal of biological chemistry, 269(45), 1994, pp. 27889-27893
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.