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 isola ted from a lambda gt11 cDNA library constructed from poly(A)(+) RNA ex tracted from etiolated cotyledons, The inserted cDNA clone contains 23 13 nucleotides and encodes a polypeptide of 690 amino acids, Analysis of the amino-terminal sequence of the protein indicates that the pumpk in acyl-CoA oxidase protein is synthesized as a larger precursor conta ining a cleavable amino-terminal presequence of 45 amino acids. This p resequence 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 pa rtial purification of ACOX from etiolated pumpkin cotyledons indicated that the ACOX cDNA codes for a long chain acyl-CoA oxidase, The amoun t of ACOX increased and reached to the maximum activity by day 5 of ge rmination 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 germinati on, increased by about 30% at day 3, and faded com pletely by day 7. T hese data indicated that the expression pattern of ACOX was very simil ar to that of the glyoxysomal enzyme 3-ketoacyl-CoA thiolase, another marker enzyme of the beta-oxidation spiral, during germination and sug gested that the expression of each enzyme of P-oxidation is coordinate ly regulated.