Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.
Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.
Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.
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D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D000460:Chlorophyta / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D014638:Vanadates; D014357:Trypsin; D006180:Proton-Translocating ATPases