A pectin-methylesterase-inhibitor-based molecular probe for in situ detection of plant pectin methylesterase activity.
In the quest of obtaining a molecular probe for in situ detection of pectin methylesterase (PME), the PME inhibitor (PMEI) was biotinylated and the biotinylated PMEI (bPMEI) was extensively characterized. Reaction conditions for single labeling of the purified PMEI with retention of its inhibitory capacity were identified. High-performance size-exclusion chromatography (HPSEC) analysis revealed that the bPMEI retained its ability to form a complex with plant PME and that it gained the capacity to strongly bind an avidin species. By means of dot-blot binding assays, the ability of the probe to recognize native and high-temperature or high-pressure denatured plant PMEs, coated on an absorptive surface, was investigated and compared to the binding characteristics of recently reported anti-PME monoclonal antibodies. Contrary to the antibodies, bPMEI only detected active PME molecules. Subsequently, both types of probes were used for PME localization in tissue-printing experiments. bPMEI proved its versatility by staining prints of carrot root, broccoli stem, and tomato fruit. Applying the tissue-printing technique on carrot roots after thermal treatment demonstrated the complementarity of bPMEI and anti-PME antibodies, with the former selectively detecting the remaining active PME and the latter staining both native and inactivated PME molecules.
Jolie
Ruben P
RP
Department of Microbial and Molecular Systems (M2S), Katholieke Universiteit Leuven, Leuven, Belgium.
Duvetter
Thomas
T
Vandevenne
Evelien
E
Van Buggenhout
Sandy
S
Van Loey
Ann M
AM
Hendrickx
Marc E
ME
D001710:Biotin / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D015335:Molecular Probes; D010944:Plants / Q000201:enzymology*
D004791:Enzyme Inhibitors; D015335:Molecular Probes; D001710:Biotin; D002265:Carboxylic Ester Hydrolases; C022323:pectinesterase