Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids

B. Papp, B. Sarkadi, A. Enyedi, A. J. Caride, J. T. Penniston, G. Gardos

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The functional domains of the in situ red cell membrane calcium pump were mapped by a double labeling technique. In inside-out vesicles (IOVs) the calcium pump was phosphorylated by [γ-32P]ATP, the proteins blotted onto nitrocellulose and tagged by monoclonal antibodies raised against the purified pump protein. After proteolytic treatment of the IOVs by trypsin, chymotrypsin, or calpain-I, the fragmentation pattern of the enzyme was followed on the double-labeled immunoblots. The changes in the kinetics of the pump were examined by parallel measurements of the active calcium uptake in IOVs. By analysis of the results of tryptic digestion, it was possible to show that the antibodies recognized three different domains of the pump: 1) a M(r) = 10,000-15,000 fragment (not seen directly) which includes the calmodulin-binding domain, 2) a nonphosphorylated M(r) = 35,000 tryptic fragment, and 3) a phosphorylated fragment of M(r) = 76,000-81,000. Chymotrypsin or calpain-I digestion of the membranes produced one major, M(r) = 125,000 fragment, which had lost antibody-binding region 1. Production of this fragment coincided with the loss of calmodulin dependence and with a calmodulin-like activation of IOV calcium uptake (high V(max), cooperativity in calcium activation). The M(r) = 125,000 fragment was further activated by acidic lipids producing high V(max) and low K( 1/2 )(Ca2+) with no cooperativity. Based on these data a kinetic model and a functional map of the plasma membrane calcium pump is suggested.

Original languageEnglish
Pages (from-to)4577-4582
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number8
Publication statusPublished - Jan 1 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Functional domains of the in situ red cell membrane calcium pump revealed by proteolysis and monoclonal antibodies. Possible sites for regulation by calpain and acidic lipids'. Together they form a unique fingerprint.

  • Cite this