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Protein farnesyltransferase: measurement of enzymatic activity in 96-well format using TopCount microplate scintillation counting technology.
Anal Biochem. 1995 Apr 10; 226(2):268-78.AB

Abstract

Protein farnesyltransferase (PFT) catalyzes the transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to proteins ending with a carboxy-terminal CAAX motif, forming a thioether linkage to the cysteine residue of the protein. A method is described herein for measurement of PFT activity in 96-well format using TopCount microplate scintillation counting technology. This method has the advantages of requiring only a single transfer from reaction vessels or wells of a 96-well reaction plate to the filtration wells of a 96-well Packard UniFilter GF/B filtration plate following acid precipitation and of allowing liquid scintillation counting to be conducted directly in the filtration plate without the need for either removal of the filter from the plate or transfer of the filter to liquid scintillation vials prior to radioactivity assessment. Using rat brain cytosol as the source of PFT, H-ras as the source of farnesyl acceptor protein, and [1-3H]FPP as the farnesyl donor, the incorporation of radiolabeled farnesyl residues into H-ras was found to be a linear function of both the time of incubation at 37 degrees C (up to 75 min) and the concentration of rat brain cytosolic protein present during incubation (up to 40 micrograms protein), and to be dependent on the concentration of H-ras (Km = 1.1 microM) and FPP (Km = 0.6 microM) present in the incubation reaction. In the presence of 4 microM H-ras, 0.5 microM FPP, 4 mM MgCl2, and 20 microM ZnCl2, the specific activity of rat brain cytosolic PFT measured using this methodology was 0.253 +/- 0.036 (SD; n = 30) pmol H-ras farnesylated per minute of incubation at 37 degrees C per milligram cytosolic protein. The signal-to-noise ratio for H-ras farnesylation using this methodology averaged 25 relative to incubation in the absence of H-ras (background farnesylation of cytosolic proteins) and 50 relative to incubation in the absence of both H-ras and rat brain cytosol (background filter associated radioactivity). Intraassay variability averaged 4% and interassay variability averaged 9%. FPP mimetics from a variety of distinct structural classes, previously shown to act as competitive inhibitors of the FPP-utilizing enzyme, squalene synthetase (SQS), also inhibited PFT activity measured using this methodology, but exhibited approximately 300- to 500-fold specificity for inhibition of SQS relative to inhibition of PFT, when both enzymes were measured at their respective Km FPP concentrations, suggesting structural differences between the FPP binding sites of the two enzymes.(

ABSTRACT

TRUNCATED AT 400 WORDS)

Authors+Show Affiliations

Department of Metabolic Diseases, Pfizer Central Research, Pfizer Inc., Groton, Connecticut 06340, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

7793628

Citation

Harwood, H J.. "Protein Farnesyltransferase: Measurement of Enzymatic Activity in 96-well Format Using TopCount Microplate Scintillation Counting Technology." Analytical Biochemistry, vol. 226, no. 2, 1995, pp. 268-78.
Harwood HJ. Protein farnesyltransferase: measurement of enzymatic activity in 96-well format using TopCount microplate scintillation counting technology. Anal Biochem. 1995;226(2):268-78.
Harwood, H. J. (1995). Protein farnesyltransferase: measurement of enzymatic activity in 96-well format using TopCount microplate scintillation counting technology. Analytical Biochemistry, 226(2), 268-78.
Harwood HJ. Protein Farnesyltransferase: Measurement of Enzymatic Activity in 96-well Format Using TopCount Microplate Scintillation Counting Technology. Anal Biochem. 1995 Apr 10;226(2):268-78. PubMed PMID: 7793628.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Protein farnesyltransferase: measurement of enzymatic activity in 96-well format using TopCount microplate scintillation counting technology. A1 - Harwood,H J,Jr PY - 1995/4/10/pubmed PY - 2001/3/28/medline PY - 1995/4/10/entrez SP - 268 EP - 78 JF - Analytical biochemistry JO - Anal Biochem VL - 226 IS - 2 N2 - Protein farnesyltransferase (PFT) catalyzes the transfer of the farnesyl group of farnesyl pyrophosphate (FPP) to proteins ending with a carboxy-terminal CAAX motif, forming a thioether linkage to the cysteine residue of the protein. A method is described herein for measurement of PFT activity in 96-well format using TopCount microplate scintillation counting technology. This method has the advantages of requiring only a single transfer from reaction vessels or wells of a 96-well reaction plate to the filtration wells of a 96-well Packard UniFilter GF/B filtration plate following acid precipitation and of allowing liquid scintillation counting to be conducted directly in the filtration plate without the need for either removal of the filter from the plate or transfer of the filter to liquid scintillation vials prior to radioactivity assessment. Using rat brain cytosol as the source of PFT, H-ras as the source of farnesyl acceptor protein, and [1-3H]FPP as the farnesyl donor, the incorporation of radiolabeled farnesyl residues into H-ras was found to be a linear function of both the time of incubation at 37 degrees C (up to 75 min) and the concentration of rat brain cytosolic protein present during incubation (up to 40 micrograms protein), and to be dependent on the concentration of H-ras (Km = 1.1 microM) and FPP (Km = 0.6 microM) present in the incubation reaction. In the presence of 4 microM H-ras, 0.5 microM FPP, 4 mM MgCl2, and 20 microM ZnCl2, the specific activity of rat brain cytosolic PFT measured using this methodology was 0.253 +/- 0.036 (SD; n = 30) pmol H-ras farnesylated per minute of incubation at 37 degrees C per milligram cytosolic protein. The signal-to-noise ratio for H-ras farnesylation using this methodology averaged 25 relative to incubation in the absence of H-ras (background farnesylation of cytosolic proteins) and 50 relative to incubation in the absence of both H-ras and rat brain cytosol (background filter associated radioactivity). Intraassay variability averaged 4% and interassay variability averaged 9%. FPP mimetics from a variety of distinct structural classes, previously shown to act as competitive inhibitors of the FPP-utilizing enzyme, squalene synthetase (SQS), also inhibited PFT activity measured using this methodology, but exhibited approximately 300- to 500-fold specificity for inhibition of SQS relative to inhibition of PFT, when both enzymes were measured at their respective Km FPP concentrations, suggesting structural differences between the FPP binding sites of the two enzymes.(ABSTRACT TRUNCATED AT 400 WORDS) SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/7793628/Protein_farnesyltransferase:_measurement_of_enzymatic_activity_in_96_well_format_using_TopCount_microplate_scintillation_counting_technology_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(85)71225-0 DB - PRIME DP - Unbound Medicine ER -