Abstract
A method is presented for the assay of starch synthase activity in plant extracts which does not require the use of radioactive substrates. The method is based on the quantification by HPLC of phosphorylated nucleosides generated in the reaction mixture during incorporation of ADP glucose (ADPGlc) into polymeric glucan. Estimation of starch synthase activity based on the rate of ADP accumulation in the medium or on the incorporation of glycosyl residues of ADP [U- 14C]Glc into polymeric produced comparable results in extracts of tobacco, spinach or potato leaves. With potato tuber extracts AMP accumulated in the medium instead of ADP. This is ascribed to the activity of apyrase (EC 3.6.1.5), a fluoride-insensitive ATP phosphohydrolase which removes g-phosphoryl groups of ATP and ADP and it is particularly abundant in potato tubers. In this case, the rate of AMP accumulation in the reaction mixture well correlated with the rate of ADP [U- 14C]Glc incorporation into polymeric glucan. Thus, the HPLC method presented for the assay of starch synthase activity is also suitable for tissues containing apyrase activity.
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