Quantification of subnanomolar amounts of phosphate bound to seryl and threonyl residues in phosphoproteins using alkaline hydrolysis and malachite green.
Ekman, P and Jäger, O
Anal. Biochem., 214: 138-41 (1993)
1993
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An assay for protein-bound phosphate with a capacity to determine 100 pmol of phosphate is described. It is based on the combination of two well known methods: the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and the quantification of the released phosphate by the use of malachite green and phosphomolybdate. | Phosphatase Assay | 8250216
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The use of phosphopeptides to distinguish between protein phosphatase and acid/alkaline phosphatase activities: opposite specificity toward phosphoseryl/phosphothreonyl substrates.
Donella-Deana, A, et al.
Biochim. Biophys. Acta, 1094: 130-3 (1991)
1991
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The four main classes of protein phosphatases (PP-1, 2A, 2B and 2C), although differing in their ability to dephosphorylate phosphopeptide substrates, invariably display a marked preference toward phosphothreonyl peptides over their phosphoseryl counterparts. Conversely, all the acidic and alkaline phosphatases tested so far dephosphorylate phosphoseryl derivatives far more readily than phosphothreonyl ones. This opposite behaviour provides a criterion for discriminating between protein dephosphorylating activity due to authentic protein phosphatases as compared to nonspecific acid and/or alkaline phosphatases. In particular the phosphothreonyl peptides RRATPVA and RRREEETPEEEAA appear to be especially suited for detecting the activity of PP-2C and PP-2A, since they are hardly dephosphorylated by acid and alkaline phosphatases. Conversely, the phosphoseryl peptides SPEEEEE and RRASPVA can provide a sensitive evaluation of the majority of acid and alkaline phosphatases, while being refractory to protein phosphatases. | | 1653021
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Inorganic and organic phosphate measurements in the nanomolar range.
Van Veldhoven, P P and Mannaerts, G P
Anal. Biochem., 161: 45-8 (1987)
1987
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A procedure, based on the complex formation of malachite green with phosphomolybdate under acidic conditions, to measure inorganic orthophosphate in the nanomolar range is described. The addition of polyvinyl alcohol is required to stabilize the dye-phosphomolybdate complex. The advantages of the assay are simplicity, stability of the reagents, and high sensitivity. Due to the high permissible acidity in the assay (0.9 N H2SO4), the method can be adapted easily to measure nanomolar amounts of phosphate, liberated from organic compounds like phosphoproteins and phospholipids after wet digestion. | Phosphatase Assay | 3578786
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