ABSTRACT
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.
Subject(s)
Alkyl and Aryl Transferases , Transferases/analysis , Transferases/metabolism , Amino Acid Sequence , Animals , Baculoviridae , Cell Line , Chromatography, High Pressure Liquid/methods , Dansyl Compounds/chemistry , Dansyl Compounds/pharmacology , Kinetics , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/pharmacology , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Spectrometry, Fluorescence/methods , Spodoptera , Substrate Specificity , Transfection , ras Proteins/metabolismABSTRACT
We describe a simple turbidometric assay for phosphatidylcholine-specific phospholipase C (PC-PLC) (EC 3.1.4.3), phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10), and sphingomyelinase (SMase) (EC 3.1.4.12), suitable for high-volume screening using unmodified substrates. Under the conditions described, 1 to 10 U/ml of PC-PLC (Bacillus cereus) induces a rapid and continuous increase in turbidity (0.4 to 0.6 AU at 410 nm) of phosphatidylcholine vesicles (1-10 mM) that highly correlates with hydrolysis. Turbidity increases with the formation of small homogenous particles, which is enzyme and substrate dependent. Analogously, PI-PLC (1-10 U/ml) causes a continuous increase in the turbidity of PI vesicles. SMase also causes a continuous increase in PC vesicle turbidity, but unlike like the glycerol phospholipases, SMase causes a discontinuous increase in vesicles of its proper substrate sphingomyelin (SM). After 8-15% hydrolysis, SM vesicles are converted to large heterogeneous particles permitting detection of SMase activity by visual inspection. Thus, turbidity is a useful property to monitor SMases and C-type phospholipases that cleave vesicle-forming phospholipids. Furthermore, the assay is designed for the microtiter plate format, enabling the continuous and simultaneous monitoring of up to 96 wells.
