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Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

S.M. Teresa Hernández-Sotomayor*, César De Los Santos-Briones, J. Armando Muñoz-Sánchez, and Victor M. Loyola-Vargas

Unidad de Biología Experimental, Centro de Investigación Científica de Yucatán, Apartado Postal 87 Cordemex 97310, Merida, Yucatan, Mexico

The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 µM. These findings will add to our understanding of the mechanisms of regulation of plant PLC.

1   This work was supported by grants from Consejo Nacional de Ciencia y Tecnológia (no. 4119P-N9609) and International Foundation for Science (no. C/2236-2), the interchange program between the Royal Society of London and the Scientific Research Academy of México, and a Consejo Nacional de Ciencia y Tecnología fellowship to C.D.L.S.-B. (no. 88202).
*   Corresponding author; e-mail ths{at}cicy.mx; fax 99-81-3900.

Plant Physiol. (1999) 120: 1075-1082
Copyright Clearance Center:   0032-0889/99/120//08
© 1999 American Society of Plant Physiologists




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