Purification of protein kinase C from bovine rod outer segments.
Rod outer segments (ROS) from bovine retinae were found to have high levels of calcium/phospholipid dependent protein kinase (protein kinase C). Protein kinase C behaves as an extrinsic membrane protein and phosphorylates rhodopsin in a calcium-dependent manner. The abundance of protein kinase C in ROS is similar to that of rhodopsin kinase. Its ability to phosphorylate rhodopsin in ROS membranes suggests protein kinase C may play an important role in the regulation of signal transduction in the ROS. The limited set of extrinsic membrane proteins and abundance of protein kinase C makes this tissue an extremely useful source to purify protein kinase C. The extrinsic membrane protein fraction has 6-7 U protein kinase C activity per mg protein, and the enzyme is quite stable apparently due to the lack of proteases in the preparation. A procedure was developed using phosphatidylserine- and calcium-dependent binding of protein kinase C to phenyl-Sepharose in low ionic strength buffer to resolve protein kinase C and other calcium-binding proteins from the majority of extrinsic membrane proteins. Protein kinase C was eluted using EGTA, and peak fractions directly loaded onto a DEAE-cellulose column. The protein kinase C peak eluted from the ion-exchange column was pooled and had a specific activity greater than 1,000 nmol phosphate transferred to histone per min per mg protein with a recovery of 25 percent of the starting activity. The procedure to purify protein kinase C from ROS is simple and can be completed in one day.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification; D010786:Photoreceptor Cells / Q000201:enzymology*; D006656:Protamine Kinase / Q000302:isolation & purification / Q000378:metabolism; D011493:Protein Kinase C / Q000302:isolation & purification* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D008565:Membrane Proteins; D006656:Protamine Kinase; D011493:Protein Kinase C; D002118:Calcium