Characterisation of Phosphorylated Intermediates Synthesised during the Catalytic Cycle of the Sodium Adenosine Triphosphatase in the Plasma Membrane of the Marine Unicellular Alga Tetraselmis (Platymonas) viridis

Abstract

Highly purified plasma membranes (PM) isolated from the marine unicellular alga Tetraselmis (Platymonas) viridis were incubated with [γ-32p]-ATP in short term labelling assays in the presence of various effectors, followed by an analysis of the PM proteins by SDS-PAGE under acidic conditions. Three radioactive components of apparent molecular masses of 100 kD, 76 kD, and 26 kD were detected. The phosphorylation of one of them, the 100 kD polypeptide, was specifically dependent on Na+ with an apparent KM of about 12 mmol/L and reached the maximal level in less than 10 s. Vanadate almost completely inhibited the phosphorylation of the peptide. The phosphate incorporated into the 100 kD polypeptide was subjected to a rapid turnover and was cleaved by hydroxylamine. The 100 kD phosphopeptide was sensitive to ADP, which caused rapid discharge of the phosphopeptide. From the data it can be deduced that the 100 kD phosphopeptide is the phosphorylated intermediate of the Na+-transporting ATPase in the T. viridis PM, the functioning of which has been demonstrated previously (Balnokin and Popova, 1994). The functional roles of the 76 kD and 26 kD peptides remain to be elucidated. On hand from the data it can be hypothesised that the 76 kD phosphopeptide may be the phosphointermediate of the H+-ATPase that has been demonstrated in the PM of T. viridis (Popova and Balnokin, 1992), while the 26 kD polypeptide may participate in the catalytic cycles of the ATPases, being a primary site of phosphate binding.