Molecular characterization of a glyoxysomal long chain acyl-CoA oxidase that is synthesized as a precursor of higher molecular mass in pumpkin.
A cDNA clone for pumpkin acyl-CoA oxidase (EC 1.3.3.6; ACOX) was isolated from a lambdagt11 cDNA library constructed from poly(A)+ RNA extracted from etiolated cotyledons. The inserted cDNA clone contains 2313 nucleotides and encodes a polypeptide of 690 amino acids. Analysis of the amino-terminal sequence of the protein indicates that the pumpkin acyl-CoA oxidase protein is synthesized as a larger precursor containing a cleavable amino-terminal presequence of 45 amino acids. This presequence shows high similarity to the typical peroxisomal targeting signal (PTS2). Western blot analysis following cell fractionation in a sucrose gradient revealed that ACOX is localized in glyoxysomes. A partial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase. The amount of ACOX increased and reached to the maximum activity by day 5 of germination but decreased about 4-fold on the following days during the subsequent microbody transition from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA was already high at day 1 of germination, increased by about 30% at day 3, and faded completely by day 7. These data indicated that the expression pattern of ACOX was very similar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the beta-oxidation spiral, during germination and suggested that the expression of each enzyme of beta-oxidation is coordinately regulated.
Hayashi
H
H
Department of Cell Biology, National Institute for Basic Biology, Okazaki, 444 Japan.
De Bellis
L
L
Yamaguchi
K
K
Kato
A
A
Hayashi
M
M
Nishimura
M
M
D042962:Acyl-CoA Oxidase; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment
D018076:DNA, Complementary; D010940:Plant Proteins; D011498:Protein Precursors; D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase