Purification and cDNA sequencing of an oleate-selective acyl-ACP:sn-glycerol-3-phosphate acyltransferase from pea chloroplasts.
The soluble acyl-ACP:sn-glycerol-3-phosphate acyltransferase from chloroplasts of chilling-sensitive and -resistant plants differ in their fatty acid selectivity. Enzymes from resistant plants discriminate against non-fluid palmitic acid and select oleic acid whereas the acyltransferase from sensitive plants accepts both fatty acids. To use this difference for improving plant chilling resistance by biotechnology the gene for an oleate-selective enzyme is required. Therefore, the oleate-selective enzyme from pea seedlings was purified to apparent homogeneity. Tryptic peptides of internal origin were sequenced. Polyclonal antibodies raised in rabbits were used for an immunological screening of a pea leaf cDNA expression library in lambda gt11. A positive clone of 1800 bp was selected showing an open reading frame which codes for 457 amino acids. The deduced amino acid sequence coincides perfectly with the tryptic sequences. A tentative assignment of the processing site was made which divides the preprotein into a mature protein of 41 kDa in accordance with experimental findings and a transit peptide of 88 amino acids. At present the comparison between a selective (pea) and an unselective (squash) acyltransferase sequence does not provide a clue for recognizing the structural differences resulting in different selectivities.
Institut für Allgemeine Botanik, Universität Hamburg, Germany.
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S
Weber
F P
FP
Wolter
F
F
Buck
M
M
Frentzen
E
E
Heinz
D008243:1-Acylglycerophosphocholine O-Acyltransferase; D000217:Acyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology*; D010449:Peptide Mapping; D010944:Plants / Q000201:enzymology / Q000235:genetics; D010946:Plants, Medicinal; D012689:Sequence Homology, Nucleic Acid
D009829:Oleic Acids; D019301:Oleic Acid; D004247:DNA; D000217:Acyltransferases; D008243:1-Acylglycerophosphocholine O-Acyltransferase