Photoinactivation of the bovine heart mitochondrial F1-ATPase by [14C]dequalinium cross-links phenylalanine-403 or phenylalanine-406 of an alpha subunit to a site or sites contained within residues 440-459 of a beta subunit.
Synthesis of [14C]dequalinium, 1,1'-(1,10-[1,10-14C]decanediyl)bis[4-amino-2-methylquinolinium ], is described, which photoinactivates the bovine heart mitochondrial F1-ATPase (MF1). Maximal photoinactivation occurs on incorporation of about 1.5 mol of [14C]dequalinium/mol of MF1. Three radioactive species were resolved when photoinactivated enzyme was submitted to polyacrylamide gel electrophoresis at pH 4.0 in the presence of tetradecyltrimethylammonium bromide, which correspond to the alpha and beta subunits and a cross-linked species with an M(r) of 116,000. Fractionation of a tryptic digest of photoinactivated enzyme by high-performance liquid chromatography led to isolation of a radioactive peptide which contains residues 399-420 of a alpha subunit. Two fragments containing equal amounts of radioactivity were obtained on fractionation of an endoproteinase Asp-N digest of the isolated radioactive tryptic peptide by high-performance liquid chromatography. Amino acid sequence analysis showed that both fragments contained residues 399-408 of the alpha subunit, but one was missing Phe-alpha 403 and the other was lacking Phe-alpha 406. Fractionation of a cyanogen bromide digest of photoinactivated enzyme followed by trypsin digestion of partially purified cyanogen bromide fragments and fractionation of the resulting radioactive tryptic fragments yielded several radioactive species comprised of residues 399-420 of the alpha subunit cross-linked to residues 440-459 of the beta subunit and a radioactive fragment containing residues 399-420 of the alpha subunit. Partial sequence analyses of the cross-linked fragments suggest that Phe-alpha 403 and Phe-alpha 406 participate in cross-links, whereas no information was obtained on the site or sites of cross-linking in the beta subunit fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhuo
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S
Department of Chemistry, University of California, San Diego, La Jolla 92093-0601.
Paik
S R
SR
Register
J A
JA
Allison
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide / Q000737:chemistry; D003868:Dequalinium / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010649:Phenylalanine / Q000737:chemistry*; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014357:Trypsin / Q000737:chemistry
D002250:Carbon Radioisotopes; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010649:Phenylalanine; D003868:Dequalinium; D014357:Trypsin; D006180:Proton-Translocating ATPases; D003488:Cyanogen Bromide