Activation and stabilization of Mg-chelatase activity by ATP as revealed by a novel in vitro continuous assay
1992
Walker, C.J. | Hupp, L.R. | Weinstein, J.D.
Mg-chelatase catalyzes the insertion of magnesium into protoporphyrin IX, which is the first step unique to chlorophyll synthesis. An organelle-free assay for Mg-chelatase has been developed from lysed pea (Pisum sativum L., cv. Spring) chloroplasts, and has now been refined by removing the bulk of the thylakoid membranes. The lighter membranes and the soluble proteins together (LM/S) have high Mg-chelatase activity (200-300 pmol Mg-deuteroporphyrin 20 min-1 mg-1 protein) and have low chlorophyll content (< 3 nmol Chl mg-1 protein), which has allowed the development of a continuous assay for Mg-chelatase. With this assay, it was shown that there was an initial lag phase of about five to six minutes before the rate of product accumulation was linear. The lag phase was considerably shortened if the LM/S were preincubated at 30 degrees C in the presence of ATP. Preincubation in the absence of ATP completely abolished Mg-chelatase activity. These data suggest that ATP is involved both in an activation process and has a stabilizing effect on Mg-chelatase activity. The details of the continuous assay are described.
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