Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

• Published in: Plant biosystems Vol. 135; no. 2; pp. 143 - 156
• Main Authors: Diffley, Penny E., Geisbrecht, Alan, Newton, Russell P., Oliver, Michael, Smith, Christopher J., Vaughan, Judith, van Cleef, Jan, van Geyschem, Jan, Walton, Terence J., Bayliss, Mark, Brenton, A. Gareth, Harris, Frank M., Games, David E., Wilkins, Adam, Roef, Luc, van Onckelen, Harry, Witters, Erwin
• Format: Journal Article
• Language: English
• Published: Taylor & Francis Group 01.01.2001
• Subjects: cations; chloroplast; cyclic-nucleotides; mass spectrometry; Phaseolus vulgaris; phosphodiesterase
• ISSN: 1126-3504; 1724-5575
• DOI: 10.1080/11263500112331350760

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li + , Na + , K + , Mg 2+ , Mn 2+ and Fe 3+ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe 3+ ions are present. Mg 2+ and Mn 2+ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe 3+ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.