Properties and kinetics of membrane-bound enzymes when both the enzyme and substrate are components of the same microsomal membrane. Studies on lathosterol 5-desaturase.
Microsomes isolated from rat liver contain an NADH-dependent lathosterol 5-desaturation system that catalyzes the introduction of a delta 5 bond into lathosterol to form 7-dehydrocholesterol. Microsomes were preloaded in vitro with liposomes composed of lathosterol and phosphatidylcholine in the presence of a high-speed supernatant (S105) protein prior to enzyme assay. The desaturation led to a reaction that occurred in two distinct phases. That is, there was an initial burst of product formation over an approximate time scale of 5 min that fell off, thereafter to a steady state rate for over 30 min. The latter steady state phase was slower than the burst phase, because lateral diffusion of the lathosterol substrate must occur before the next reaction can take place. The total amount of the burst, which may be obtained by extrapolating the linear part of the curve in the steady state phase back to zero time, provides a means of obtaining the enzyme concentration in terms of functional active sites. It was found that the kinetics between enzyme and substrate within the same membrane also followed the usual kinetic formalism of a Michaelis-Menten type reaction as in nonaggregated homogenous solution.
Department of Biochemistry, Hokkaido University, School of Medicine, Japan.
Y
Y
Takakuwa
H
H
Nishino
Y
Y
Ishibe
T
T
Ishibashi
D000818:Animals; D002784:Cholesterol / Q000302:isolation & purification / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003684:Dehydrocholesterols / Q000302:isolation & purification / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D010713:Phosphatidylcholines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D003684:Dehydrocholesterols; D008081:Liposomes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; C033144:lathosterol delta-5-dehydrogenase; C001521:lathosterol; D002784:Cholesterol; C016705:7-dehydrocholesterol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors