Chromatographic assay and peptide substrate characterization of partially purified farnesyl- and geranylgeranyltransferases from rat brain cytosol.
A simple method for partially purifying both farnesyltransferase and geranylgeranyltransferase from rat brain cytosol is presented. Each of the final protein preparations contains one single transferase activity. A common method of measurement of both activities is described. The assay, which follows substrate prenylation, is also convenient for the measurement of the concomitant decrease in cosubstrates during the two transfer reactions. The quantitative HPLC detection of the prenylated substrates and of the cosubstrate consumption is used here to follow the purification processes. The same method is also used for substrate-specificity studies of the two enzymes performed on 18 synthetic hexapeptides derived from the C-terminus of proteins known to be prenylated in vivo. These studies partially confirm the reported differences in the substrate specificities of the two prenyltransferases. However, the observed recognition of overlapping sequences by the two enzymes might have important consequences for the inhibition of either of the enzymes in vivo and for the design of specific inhibitors.
Boutin
J A
JA
Department of Peptide and Combinatorial Chemistry, Institut de Recherches Servier, Suresnes, France. jaboutin@servier.fr
Marande
W
W
Goussard
M
M
Loynel
A
A
Canet
E
E
Fauchere
J L
JL
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology*; D051231:Farnesyltranstransferase; D009842:Oligopeptides / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D009842:Oligopeptides; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase