TY - JOUR
T1 - A novel accessory subunit for vacuolar H+-ATPase from chromaffin granules
AU - Supek, Frantisek
AU - Supekova, Lubica
AU - Mandiyan, Sreekala
AU - Pan, Yu Ching E.
AU - Nelson, Hannah
AU - Nelson, Nathan
PY - 1994/9/30
Y1 - 1994/9/30
N2 - Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.
AB - Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.
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AN - SCOPUS:0028067756
SN - 0021-9258
VL - 269
SP - 24102
EP - 24106
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 39
ER -