Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome-b5 reductase systems in phosphatidylcholine vesicles.
The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeled cytochrome b5 (DNS-cytochrome b5), on interaction with NADH-cytochrome-b5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15-30 degrees C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15-30 degrees C. The enzyme activity of the DNS-cytochrome-b5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b5 and NADH-cytochrome-b5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.
Pugh
E L
EL
Department of Biochemistry, University of Ottawa, Canada.
Kates
M
M
Szabo
A G
AG
Krajcarski
D T
DT
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism*; D003619:Dansyl Compounds; D005454:Fluorescence Polarization; D007202:Indicators and Reagents; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism
D003619:Dansyl Compounds; D007202:Indicators and Reagents; D010713:Phosphatidylcholines; D009243:NAD; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase