Kinetic analysis of phospholipase C from Catharanthus roseus transformed roots using different assays

• Published in: Plant physiology (Bethesda) Vol. 120; no. 4; pp. 1075 - 1081
• Main Authors: Hernandez-Sotomayor, S.M, Santos-Briones, C. de los, Munoz-Sanchez, J.A, Loyola-Vargas, V.M
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01.08.1999; American Society of Plant Biologists
• Subjects: Agronomy. Soil science and plant productions; assays; binding; Biological and medical sciences; Catharanthus roseus; Cell Biology and Signal Transduction; chemical constituents of plants; Economic plant physiology; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; genetic transformation; Hydrolysis; isotope labeling; Kinetics; Lipids; Metabolism; Metabolism. Physicochemical requirements; Micelles; Nitrogen metabolism and other ones (excepting carbon metabolism); Nutrition. Photosynthesis. Respiration. Metabolism; phospholipase C; Phospholipids; Plant physiology and development; Plant roots; Plants; Pressure; Protein isoforms; roots; tissue culture; transgenic plants; vesicles; Purification; Root; Enzyme; Isozyme; Esterases; Catharanthus roseus; Biomembrane; Phosphoric diester hydrolases; Medicinal plant; Phospholipase C; Apocynaceae; Characterization; Dicotyledones; Angiospermae; Hydrolases; Spermatophyta; Transgenic plant; Kinetics; Catalyst activity; Anchoring
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.120.4.1075

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 micromolar. These findings will add to our understanding of the mechanisms of regulation of plant PLC.