Affinity of synthetic peptides for the HSV-2 ribonucleotide reductase R1 subunit measured with an iodinated photoaffinity peptide.
The herpes simplex virus (HSV) ribonucleotide reductase comprises two nonidentical subunits, R1 and R2, which associate to form the active holoenzyme. A sensitive binding assay was developed to measure the affinity of inhibitory peptides for the HSV R1 subunit. The assay involved the use of a photoreactive radioligand [4'-azido-Phe328,3',5'-125I-Tyr329] HSV R2-(328-337), an analogue of the decapeptide Ser-Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu which corresponds to the C-terminal sequence (328-337) of the HSV R2 protein. As the radioligand binds covalently to the HSV R1 subunit upon uv irradiation, the affinity of peptide inhibitors can be easily determined by measuring their ability to compete with this highly specific binding. The method, which did not require any pure preparation of R1, was tested at 25 and 4 degrees C and showed a significant increase in the affinity of the peptide inhibitors at 4 degrees C. The relative affinity of these peptides was in agreement with their relative potency to inhibit reductase activity. The affinity of R2 subunit for R1 was also determined, and an IC50 of 0.05 microM was measured. Altogether, this assay represents a precise and reliable tool with which to study more potent HSV ribonucleotide reductase peptide inhibitors, and the method could be applied to the study of other protein-protein and peptide-protein interactions.
Institut du Cancer de Montréal, Notre-Dame Hospital, Quebec, Canada.
N
N
Lamarche
P
P
Gaudreau
B
B
Massie
Y
Y
Langelier
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D018258:Herpesvirus 2, Human / Q000201:enzymology*; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000528:radiation effects; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000528:radiation effects; D012680:Sensitivity and Specificity; D014466:Ultraviolet Rays
D000345:Affinity Labels; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D009842:Oligopeptides; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases