Purification of a trypsin inhibitor (PFTI) from pumpkin fruit phloem exudate and isolation of putative trypsin and chymotrypsin inhibitor cDNA clones.
The major trypsin inhibitor from pumpkin (Cucurbita maxima cv Supermarket Hybrid) fruit phloem exudate was purified by affinity and reverse phase chromatography. The protein has a molecular weight of approximately 8100 by SDS-PAGE and is blocked at the N-terminal serine. Following sequencing of a CNBr fragment, 3'- and 5'-RACE were used to isolate full length cDNAs corresponding to a trypsin inhibitor and to two chymotrypsin inhibitors. The three genes are similar, both in their translated and non-translated regions. Comparison of the full length translated proteins show that they are members of the proteinase inhibitor I family and almost identical apart from the P1 site in the proteinase binding loop. The genes encode proteins of 67 amino acids and appear to lack not only both pre- and prepro-peptide sequences but also the single disulphide present in most proteinase inhibitor I family members.
Murray
C
C
Pest Resistance Group, Horticulture and Food Research Institute of New Zealand, Palmerston North.
Christeller
J T
JT
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000037:antagonists & inhibitors*; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005122:Exudates and Transudates / Q000737:chemistry*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010944:Plants / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification*
D018744:DNA, Plant; D015345:Oligonucleotide Probes; D014361:Trypsin Inhibitors; D002918:Chymotrypsin