A simple determination of the sideness of the NH2-terminus in the membrane bound cytochrome P-450 LM2.
A simple and fast procedure for determining the sideness of the NH2-terminus in membrane proteins using FITC as label has been developed and applied to microsomal cytochrome P-450 LM2. The main advantages of FITC, which was shown not to penetrate lipid membranes, as compared to other labels are that it is fluorescent and that it can be used for manual microsequencing of proteins. By use of FITC it was directly shown that the NH2-terminus of P-450 LM2 is localized at the cytoplasmic side of the endoplasmic reticulum membrane. Based on this result the validity of recently published models of the membrane topology and the membrane insertion of P-450 are discussed.
Bernhardt
R
R
Central Institute of Molecular Biology, Academy of Sciences, Berlin-Buch, GDR.
Kraft
R
R
Ruckpaul
K
K
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D008715:Methionine / Q000032:analysis; D008862:Microsomes, Liver / Q000201:enzymology; D011817:Rabbits; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D013861:Thiocyanates; D003577:Cytochrome P-450 Enzyme System; D008715:Methionine; D016650:Fluorescein-5-isothiocyanate