Phosphorylation of the Ca2+ pump intermediate in intact red cells, isolated membranes and inside-out vesicles

• Published in: Molecular and cellular biochemistry Vol. 22; no. 2-3; p. 147
• Main Authors: Szász, I, Hasitz, M, Sarkadi, B, Gárdos, G
• Format: Journal Article
• Language: English
• Published: Netherlands 22.12.1978
• Subjects: Biological Transport, Active - drug effects; Calcium - blood; Calcium - pharmacology; Erythrocyte Membrane - drug effects; Erythrocyte Membrane - metabolism; Erythrocytes - drug effects; Erythrocytes - metabolism; Humans; Lanthanum - pharmacology; Membrane Proteins - blood; Mercury - pharmacology; Phosphoproteins - blood; Phosphorylation
• ISSN: 0300-8177
• DOI: 10.1007/BF00496240

Ca2+-entry into intact red cells containing [32P]-ATP increases the phosphorylation of the 150 000 dalton polypeptide of the membrane. This phosphorylation occurs even in Mg2+-depleted red cells. Extracellular lanthanum applied during ATP-depletion further increases the Ca2+-induced phosphorylation. In fragmented membranes or resealed insideout vesicles (IOVs) membrane bound Mg2+ is sufficient to catalyze the phosphorylation of spectrin 2 and Band 3 polypeptides with low concentrations (less than micron of [32P]-ATP. In Ca-EDTA buffers one single polypeptide is phosphorylated which is located in the 150 000 molecular weight region. KmCa for phosphorylation is much lower (0.2 micron) than for active Ca2+ transport (40 micron) in IOVs. Lanthanum induced phosphorylation (up to 250 micron Lafree) is significantly greater than Ca2+-induced phosphorylation. Hg2+ inhibits both Ca2+ and La3+ induced phosphorylation. Ca2+-induced labelling can be rapidly "chased" by unlabelled ATP+Mg2+, but not with EGTA+Mg2+. Dephosphorylation in Ca2+ phosphorylated membranes and IOVs is significantly inhibited by La3+. It can be concluded that the mechanism of La3+ and Hg2+ inhibition of the Ca2+ pump is different in intact cells and isolated membranes or Iovs.