Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney

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Abstract
Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (s20,w) of .apprx. 7.4 S and a MW of .apprx. 150,000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of 2 subunits with MW of .apprx. 97,000 and 50,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatase activity resides in the MW 50,000 subunit, which is sensitive to proteolysis. The phosphatase contains .apprx. 1 mol of FAD/mol of protein of MW 150,000. FAD is apparently associated with the MW 97,000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+/mol of enzyme of MW 150,000 at pH 7.0, with a Kd of .apprx. 35 .mu.M as determined by flow dialysis. Use of ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of .apprx. 8 .mu.M. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, 2 equivalent and apparently noninteracting Ca2+-binding sites were detected per unit of MW 150,000, with a Kd value of .apprx. 24 .mu.M in the absence and .apprx. 5 .mu.M in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity .apprx. 95%, the phosphatase exhibited only 1 Ca2-binding sites, even in the presence of E2. The phosphatase apparently possesses an intrinsic Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second is apparently altered by increasing the ionic strength. The second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.