ABSTRACT
Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.
Subject(s)
Chromaffin Granules/enzymology , Membrane Proteins/metabolism , Proton-Translocating ATPases/metabolism , Vacuolar Proton-Translocating ATPases , Amino Acid Sequence , Animals , Base Sequence , Cattle , Humans , Membrane Proteins/chemistry , Mice , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
Glycoprotein IV of bovine adrenal chromaffin granule membranes was purified by membrane fractionation with Triton X-114 and lectin affinity chromatography. An antiserum raised against this protein recognized the same component as one directed against subunit Ac45 of the proton-translocating adenosine triphosphatase in the granule membrane. Amino acid sequencing confirmed that glycoprotein IV and Ac45 are identical proteins, and also showed that they are derived from a larger precursor by removal of a 246-amino acid N-terminal sequence. Enzymatic deglycosylation indicated an apparent polypeptide molecular mass of 29 kDa for the mature Ac45/glycoprotein IV. Blue Native electrophoresis confirmed that this protein is a component of the membrane sector of the V-ATPase.
