Characterization of Xenopus laevis oocyte farnesyl transferase.
Farnesyl transferase (FTase) activity, characterized in extracts of Xenopus laevis oocytes using an in vitro filtration assay, was observed to be at least 80% cytosolic with optimal product formation at pH 7.0. Oocyte FTase displayed enzyme activity that was specific for the Ras-CVIM construct but not Ras-CAIL or Ras-SVLS. Labeling of Ras-CVIM using [3H]farnesyl pyrophosphate was inhibited by addition of unlabeled farnesyl pyrophosphate to the assay mixture but not by addition of either geranylgeranyl pyrophosphate or stearic acid. Polyclonal rabbit antibodies against recombinant human FTase subunits were tested for cross-reactivity with oocyte proteins and were used to determine the apparent molecular masses of oocyte FTase enzyme subunits. Anti-alpha subunit antibodies labeled a band of approximately 53 kDa. Anti-beta subunit antibodies bound both a high molecular mass band (140 kDa) and a lower molecular mass band (38 kDa) on immunoblots of cytosolic oocyte proteins. When oocyte FTase activity was resolved by gel filtration, the peak of enzyme activity correlated with immunoblot detection of the 38- and 53-kDa bands as well as a doublet of 86- and 95-kDa. This correlation suggests that Xenopus laevis oocyte FTase is a heterodimer under reducing conditions with subunits of 53 +/- 1 and 38 +/- 2 kDa, and that the holoenzyme migrates on SDS-polyacrylamide gels with an apparent molecular mass of 86-95 kDa.
Goalstone
M L
ML
Department of Biological Sciences, University of Denver, Colorado 80208, USA.
Diamond
C L
CL
Sadler
S E
SE
D019883:Alkyl and Aryl Transferases; D000818:Animals; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D051231:Farnesyltranstransferase; D005260:Female; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D046911:Macromolecular Substances; D008970:Molecular Weight; D009865:Oocytes / Q000201:enzymology* / Q000648:ultrastructure; D011106:Polyisoprenyl Phosphates / Q000378:metabolism / Q000494:pharmacology; D012717:Sesquiterpenes; D014166:Transferases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014982:Xenopus laevis; D018631:ras Proteins / Q000378:metabolism
D046911:Macromolecular Substances; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase; D018631:ras Proteins