A fluorescence assay for geranylgeranyl transferase type I.
A new fluorescence assay for measuring the activity of geranylgeranyl transferase (type I) is described. It does not require the use of either radiolabeled geranylgeranyl diphosphate or the purified recombinant Ras protein substrate with the carboxy terminal sequence of CVLL. Dansyl GCVLL and unlabeled geranylgeranyl diphosphate are used as substrates. The Km for Dansyl GCVLL and for geranylgeranyl diphosphate is 5 microM and 800 nM, respectively. At equimolar concentrations, enzymatic activity is higher when Dansyl GCVLL is used as a substrate compared to Dansyl GCVII. Dansyl GCVLS, a substrate for farnesyl transferase, is inactive in this assay. CVFL is a competitive inhibitor of geranylgeranyl transferase and exhibits a Ki of 200 nM.
Pickett
W C
WC
Oncology and Immunology Research Section, American Cyanamid Company, Lederle Laboratories, Pearl River, New York 10965, USA.
Zhang
F L
FL
Silverstrim
C
C
Schow
S R
SR
Wick
M M
MM
Kerwar
S S
SS
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003619:Dansyl Compounds / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection; D014166:Transferases / Q000032:analysis* / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism
D003619:Dansyl Compounds; D009842:Oligopeptides; D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D018631:ras Proteins