A glycoprotein inhibitor of pectin methylesterase in kiwi fruit. Purification by affinity chromatography and evidence of a ripening-related precursor.
The pectin methylesterase inhibitor from kiwi fruit (Actinidia chinensis) was purified by a single-step procedure based on affinity chromatography. Partially purified tomato pectin methylesterase was covalently bound to Sepharose. The affinity resin strongly and selectively binds the inhibitor, which could be eluted in high yield as a single, homogeneous and sharp peak by high salt concentration at pH 9.5 without loss of inhibitory activity. The purified protein possesses a molecular mass of 18 kDa, as estimated by SDS/PAGE, whereas by gel filtration under native conditions, its molecular mass appears to be 25 kDa. The inhibitor interacts with pectin methylesterase, forming a 1:1 complex, as demonstrated by gel-filtration experiments. The inhibitor was glycosylated. Its glycidic portion can be removed by digestion with N-glycosidase F after protein denaturation and, to a minor extent, by digestion with N-glycosidase H. No glycidic residue could be removed by digesting the native protein with those N-glycosidases. Antibodies against pectin methylesterase inhibitor were raised in rabbits and used to evidence protein expression during fruit ripening. The results showed that the inhibitor is present in the unripe fruit as an inactive precursor with a higher molecular mass (30 kDa) and is transformed into the active protein, most likely by proteinase action, during the course of the ripening process.
Department of Biochemistry and Biophysics, II University of Naples, Italy.
A
A
Giovane
C
C
Balestrieri
L
L
Quagliuolo
D
D
Castaldo
L
L
Servillo
D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors*; D002846:Chromatography, Affinity; D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D005638:Fruit / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification* / Q000378:metabolism
D004791:Enzyme Inhibitors; D006023:Glycoproteins; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase