Transmembrane topography of the mitochondrial phosphate carrier explored by peptide-specific antibodies and enzymatic digestion.
Two peptides corresponding to the amino acid sequences 1-10 (N-terminal peptide) and 303-313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide, they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr approximately equal to 14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mr approximately equal to 17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152.(ABSTRACT TRUNCATED AT 250 WORDS)
Capobianco
L
L
Department of Pharmaco-Biology, University of Bari, Italy.
Brandolin
G
G
Palmieri
F
F
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002417:Cattle; D007106:Immune Sera; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D008929:Mitochondria, Heart / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D028044:Phosphate-Binding Proteins; D011487:Protein Conformation; D013367:Submitochondrial Particles / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D002352:Carrier Proteins; D007106:Immune Sera; D008565:Membrane Proteins; D009842:Oligopeptides; D028044:Phosphate-Binding Proteins