Sequence analysis of a proteolyzed site in Drosophila choline acetyltransferase.
The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities.
Slemmon
J R
JR
Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, NY 14642.
D000595:Amino Acid Sequence; D000818:Animals; D002795:Choline O-Acetyltransferase / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA; D004330:Drosophila / Q000201:enzymology*; D006868:Hydrolysis; D008970:Molecular Weight; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D011506:Proteins / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013552:Swine / Q000378:metabolism
D010455:Peptides; D011506:Proteins; D004247:DNA; D002795:Choline O-Acetyltransferase