The amino terminus of the putative Drosophila choline acetyltransferase precursor is cleaved to yield the 67 kDa enzyme.
A putative precursor of the 67 kDa choline acetyltransferase (Acetyl-CoA: choline-O-acetyltransferase; EC 2.3.1.6) polypeptide from Drosophila was examined using polyclonal antibodies. The central purpose of the study was to probe the suspected precursor with anti-peptide antibodies that could identify a cleavable amino terminal domain, since such a structure could be responsible for targeting the enzyme to the presynaptic terminal. Antisera were produced to both a plasmid-expressed fusion-free enzyme protein and a 26-amino acid-long peptide reproducing sequence from the enzyme. Both antisera were capable of precipitating enzyme activity from crude supernatants. Western blotting with the antibody to the plasmid-expressed enzyme visualized a major polypeptide at 75 kDa and minor polypeptides at 67 and 54 kDa. Affinity-purified IgG to the synthetic peptide only recognized the 75 kDa component and was unable to recognize purified 67 kDa enzyme protein. Timed autolysis of the enzyme in crude homogenates demonstrated both a 67 kDa polypeptide that was present prior to homogenization and a species that appeared as a product of the autolysis. The evidence from this study is consistent with the expectation that the 75 kDa band, visualized on Western blots with antisera to the enzyme, is an authentic enzyme protein. These data further suggested that the 75 kDa protein is an amino-terminally extended precursor of the 67 kDa enzyme that can be cleaved to generate the 67 kDa species.
Slemmon
J R
JR
Department of Biochemistry, University of Rochester, School of Medicine and Dentistry, NY 14642.
Campbell
G A
GA
Selski
D J
DJ
Bramson
H N
HN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002795:Choline O-Acetyltransferase / Q000737:chemistry*; D004330:Drosophila / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry*; D007074:Immunoglobulin G / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011233:Precipitin Tests; D011480:Protease Inhibitors / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D007074:Immunoglobulin G; D011480:Protease Inhibitors; D002795:Choline O-Acetyltransferase