Effects of Anoxia on Protein Phosphatase in Turtle Organs: Purification and Properties of Protein Phosphatase Type-1 from Turtle Liver

Abstract

Protein phosphatase type 1 (PP-1) was analyzed in organs of the red-eared slider turtle, Trachemys scripta elegans, a species capable of long-term anoxia survival. During anoxic submergence at 7°C, PP-1 activity in liver rapidly decreased to 63% of the control value within the first hour and remained suppressed over the subsequent 20 h of anoxia. PP-1 activity was also suppressed in red skeletal muscle during anoxia and dropped transiently (after 1 h) in brain but did not change in heart or white muscle. PP-1 was purified from turtle liver using polyethylene glycol fractionation and chromatography on DEAE-cellulose, blue dextran, Sephacryl S-200, and ADP-agarose. A 3000-fold purification was achieved with a final specific activity of 3156 nmol released min−1 mg protein−1 using 32P-labeled phosphorylase a as the substrate. Turtle liver PP-1 was a monomer of molecular mass 37 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or 38 ± 2 kDa by Sephacryl S-200 gel filtration. The enzyme was inhibited by okadaic acid (Ki 12.6 ± 1.4 nM) and AMP (Ki 23 ± 2 μM) as well as by ADP, ATP, and IMP. Regulation of liver PP-1 appears to be an integral part of anoxia-induced changes in liver glycogenolysis and metabolic rate suppression.