Properties of Kaurene Synthetase from Marah macrocarpus

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Abstract
The kaurene synthetase from immature seeds of Marah macrocarpus (Greene) Greene was partially purified from cell-free homogenates of endosperm by a combination of QAE-Sephadex A-25 chromatography and hydroxyapatite chromatography and freed of contaminating phosphatase activity. The two catalytic activities associated with kaurene synthetase, the cyclization of geranylgeranyl-pyrophosphate to copalyl-pyrophosphate (activity A) and the cyclization of copalyl-pyrophosphate to ent-kaurene (activity B), were not even partially resolved from one another during these procedures. Both activities had identical elution profiles from a calibrated Sepharose 4B column corresponding to a molecular weight less than that of ovalbumin (45,000). The A and B activities had pH optima of 7.3 and 6.9, respectively. Both activities required millimolar concentrations of the following divalent cations in the order: Mg2+ > Mn2+ > Co2+. Activities A and B were both sensitive to inhibition by Hg2+, Cu2+, p-hydroxymercuribenzoate, and N-ethylmaleimide, but activity B was much more sensitive than activity A. The average value of Km′ (apparent Km in the absence of substrate inhibition) for geranylgeranyl-pyrophosphate was 1.6 μm. Values of 0.5 and 0.6 μm were obtained for Km′ and Km, respectively, for copalyl-pyrophosphate. The Vm′ values for the two activities were similar: 12 and 9 pmol/minute·μg protein for activities A and B, respectively. N,N-Dimethylaminoethyl-2,2-diphenylpentanoate (SKF-525A) and N,N-dimethylaminoethyl-2,2-diphenylphentyl ether (SKF-3301A), tributyl-2,4-dichlorobenzylphosphonium chloride (Phosfon D), tributyl-2,4-dichlorobenzylammonium chloride (Phosfon S), 2′-isopropyl-4′-(trimethylammonium chloride)-5′-methylphenyl piperidine-1-carboxylate (Amo-1618), 2-(N,N-dimethyl-N-heptylammonium bromide)-p-methan-1-ol (Q-58), and 2-(N,N-dimethyl-N-octylammonium bromide)-p-methan-1-ol (Q-64), at concentrations from 1 to 5 μm, were effective inhibitors of kaurene synthetase activity A. Acetylcholine chloride and 2-chloroethyl-trimethylammonium chloride were effective inhibitors of activity A only at concentrations of 5 mm or greater. Abscisic acid, indole-3-acetate, gibberellin A1, gibberellin A3, a mixture of gibberellins A4 and A7, gibberellin A13, and N,N-dimethylaminosuccinamic acid (B995) were not inhibitory at any of the levels tested. None of these compounds was an effective inhibitor of activity B at concentrations less than 0.5 mm.