Purification and properties of carnitine acetyltransferase from human liver.
Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.
Bloisi
W
W
Instituto Nazionale Neurologico C. Besta, Dipartimento di Biochimica e Genetica, Milano, Italy.
Colombo
I
I
Garavaglia
B
B
Giardini
R
R
Finocchiaro
G
G
Didonato
S
S
D000123:Acetyltransferases / Q000302:isolation & purification*; D001921:Brain / Q000201:enzymology; D002332:Carnitine O-Acetyltransferase / Q000276:immunology / Q000302:isolation & purification*; D002384:Catalysis; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis / Q000276:immunology; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology; D011233:Precipitin Tests
D007106:Immune Sera; D000123:Acetyltransferases; D002332:Carnitine O-Acetyltransferase