Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity.
Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.
Paradis
H
H
Institut du Cancer de Montréal, Québec, Canada.
Gaudreau
P
P
Massie
B
B
Lamarche
N
N
Guilbault
C
C
Gravel
S
S
Langelier
Y
Y
D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000166:cytology / Q000382:microbiology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012264:Ribonucleotide Reductases / Q000302:isolation & purification* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology*
D012264:Ribonucleotide Reductases; D011494:Protein Kinases