<b>EVIDENCE OF <i>IN VIVO</i> PHOSPHORYLATION OF ERYTHROCYTE AND LIVER PYRUVATE </b><b>KINASES </b>

Abstract

Rat red cells and liver were obtained 4 hr after intravenous injection of ortho[32P]-phosphate. Erythrocyte pyruvate kinase (PK; EC 2.7.1.40) and L-type PK were recovered by the double antibody technique and applied to SDS-polyacrylamide gel electrophoresis. Incorporation of "P into erythrocyte PK and L-type PK was detected on autoradiograms of the electrophoresis corresponding to the L' and L subunits, respectively. The results demonstrated directly in viva phosphorylation of rat erythrocyte and L-type PKs.Rat and human erythrocyte PKs of the partially purified samples were phosphorylated using [T-32P]ATP. These phosphorylated PKs labelled with "P were incubated with MgCl2, resulting in the dephosphorylation and reactivation ofthe PKs, which indicate in viva dephosphorylation by the endogenous phosphoprotein phosphatase. Under the identical experimental conditions, samples freshly prepared from human red cells and human liver incubated with MgCl, resulted in the elevation of PK activity, indicating that human erythrocyte and L-type PKs are dephosphorylated by endogenous phosphatase in the presence of MgCl2. These results present indirect evidence that human erythrocyte a.nd L-type PKs are regulated in viva by the phosphorylation-dephosphorylation process.KEY WORDS phosphorylation / erythrocyte pyruvate kinase / L-type pyruvate kinase Erythrocyte pyruvate kinase (PK; EC 2.7.1.40) and L-type PK are phosphorylated by cyclic AMP-dependent protein kinase, and phosphorylated PKs exhibit a lower affinity for phosphoenolpyruvate (PEP) than unphosphorylated PKs in virra (2-9, ll, l2). The reactivation of both erythrocyte and L-type PKs by dephosphorylation has also been reported in virra (5, ll). These observations suggest that PK activity may be regulated in viva by a phosphorylation-dephosphorylation process. L-type PK was shown to be phosphorylated in viva in the rat (10), but there has been no evidence that the phosphorylation of erythrocyte PK really takes place in viva.This paper presents direct evidence of in viva phosphorylation of rat erythrocyte and L-type PKs and indirect evidence of in viva phosphorylation of human erythrocyte and L-type PKs. In addition, the dephosphorylation of erythrocyte PK by endogenous phosphatase was demonstrated, though dephosphorylation of erythrocyte PK was reported using exogenous phosphatase (5). MATERIALS AND METHODSAssay and SDS-Pa/yacrylamide Gel Electrophoresis afPK PK was assayed as reported previously (4) without the addition of ATP in the assay medium. SDS-polyacrylamide slab gel electrophoresis was performed according to Fairbanks er al. (1).

<b>CALMODULIN STIMULATES DEPHOSPHORYLATION OF A 90 kDa PROTEIN IN THE HUMAN INSULINOMA </b><b>CYTOSOL </b>

“…After incubation for various minutes at 30°C, the reaction was terminated by the addition of 25 ,ul of a solution containing 0.313 M Tris-HCI buffer (pH 6.7), 50% glycerol, 10% sodium dodecyl sulfate (SDS), 5 % 2-mercaptoethanol and 0.1 'X, phenol red. Samples were then subjected to SDS-polyacrylamide gel electrophoresis, and the dried gels were exposed to X-ray films for two weeks (3,4). The autoradiograms were scanned with a linear transport accessory attached to a spectrophotometer at a wave length of 550 nm.…”