Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins.
The purification and functional characterization of protein kinase A catalytic subunit (PKAcat) from bovine lens cytosol has been described. Purification to homogeneity has been achieved by using 100 kDa cut-off membrane filtration followed by Sephacryl S-300 chromatography and finally fractionating on High Q anion exchange column. The purified protein migrates as a single band of molecular mass approximately 41 kDa on 12.5% SDS-PAGE. Proteomic data from ion trap LC-MS when analyzed through NCBI blast program reveals significant homology (52%) with bovine zeta-crystallin and also some homology with pig casein kinase I alpha chain (38%) and SLA-DR1 beta 1 domain (38%). The search does not indicate homology with any known catalytic subunit of PKA. Inspite of the significant homology with the zeta-crystallin, our protein is different from it in terms of molecular mass. pI value of the kinase (5.3) obtained from 2D analysis is also different from zeta-crystallin (8.5). The protein is found to contain 17% alpha-helix, 26.5% beta-sheet, 21.4% turn and 34.7% random coil. The active catalytic subunit of the bovine lens cAMP-dependent kinase belongs to Type I Calpha subtype. The enzyme shows maximum activity at 30 min incubation in presence of 5 mM MgCl(2 )and 50 microM ATP. The kinase shows broad substrate specificity. It prefers Ser over Thr as phosphorylating residue. Phosphorylation of crystallin proteins, major protein fraction of bovine lens and phosphorylation of chaperone protein alpha crystallin by the kinase suggests that the kinase plays some crucial role in regulation of chaperone function within lens.
Samanta
Bhaswati
B
Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Kolkata 700009, India.
Nagdas
Subir Kumar
SK
Das
Kalipada
K
Sen
Parimal Chandra
PC
D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism*; D007908:Lens, Crystalline / Q000737:chemistry*; D010766:Phosphorylation; D011817:Rabbits
D003459:Crystallins; D017868:Cyclic AMP-Dependent Protein Kinases