Purification and Characterization of Farnesyl Protein Transferase from Bovine Testis

• Published in: BMB reports Vol. 28; no. 3; pp. 197 - 203
• Main Authors: Kwon Yul Ryu, Young Jin Baik, Chul Hak Yang
• Format: Journal Article
• Language: Korean
• Published: 생화학분자생물학회 30.05.1995
• ISSN: 1976-6696

Famesyl protein transferase involved in the first step of post-translational modification of p21^(ras) proteins transfers the famesyl moiety from famesyl pyrophosphate to a cysteine residue in p21^(ras) proteins. The enzyme was first purified 30,000-fold from bovine testis by use of 30∼50% ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, Sephacryl S-300 gel filtration chromatography, Sephacryl S-200 gel filtration chromatography, and hexapeptide (Lys-Lys-Cys-Val-Ile-Met) affinity chromatography. The molecular weight of the purified enzyme was estimated to be ∼100 kDa by gel filtration and SDS-polyacrylamide gels showed two closely spaced bands of ∼50 kDa protein. These indicate that the enryme consists of two nonidentical subunits, a and β, which have slightly different molecular weights. The enzyme was inhibited by hexapeptide (Lys-Lys-Cys-Val-Ile-Met), which acted as an alternative substrate that competed for famesylation. Kinetic analysis by measuring initial velocities showed that famesyl protein transferase is a very slow enzyme. EDTA-treated famesyl protein transferase showed little activity with Mg^(2+) or Zn^(2+) alone, but required both Mg^(2+) and Zn^(2+) for the catalytic activity.