A malachite green-based assay to assess glucan phosphatase activity

Abstract

With the recent discovery of a unique class of dual-specificity phosphatases that dephosphorylate glucans, we report an in vitro assay tailored for the detection of phosphatase activity against phosphorylated glucans. We demonstrate that in contrast to a general phosphatase assay utilizing a synthetic substrate, only phosphatases that possess glucan phosphatase activity liberate phosphate from the phosphorylated glucan amylopectin using the described assay. This assay is simple and cost-effective, providing reproducible results that clearly establish the presence or absence of glucan phosphatase activity. The assay described will be a useful tool in characterizing emerging members of the glucan phosphatase family.

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

“…SEX4 (0.88 Ϯ 0.08 nmol P i released ϫ min Ϫ1 ϫ nmol protein Ϫ1 ), LSF2 (0.22 Ϯ 0.01 nmol P i released ϫ min Ϫ1 ϫ nmol protein Ϫ1 ), and laforin (10.6 Ϯ 0.4 nmol P i released ϫ min Ϫ1 ϫ nmol protein Ϫ1 ), all displayed robust specific activity (Fig. 4, A-C), consistent with previous studies (61).…”

“…Phosphatase Assays-Generic phosphatase activity assays were performed using para-nitrophenyl phosphate (pNPP) (31,34,61). Hydrolysis of pNPP was performed in 50 l reactions, containing 1x phosphatase buffer (100 mM sodium acetate, 50 mM bis-Tris, 50 mM Tris-HCl, pH 7.0), 2 mM DTT, and 50 mM pNPP.…”

“…Glucan phosphatase activity assays were performed with amylopectin using malachite green (31,34,61). Reactions were performed in 20 l containing 1x phosphatase buffer (100 mM sodium acetate, 50 mM bus-Tris, 50 mM Tris-HCl, pH 7.0) and 2 mM DTT, and 45 g amylopectin, and 150 ng protein.…”

Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease

“…Laforin is the only known human phosphatase that can dephosphorylate glucan substrates (Gentry et al, 2007; Sherwood et al, 2013; Worby et al, 2006). To confirm the function of the crystallized laforin construct, we tested the ability of the catalytically active protein to both bind and dephosphorylate glycogen.…”

Structure of the Arabidopsis Glucan Phosphatase LIKE SEX FOUR2 Reveals a Unique Mechanism for Starch Dephosphorylation

“…Phosphatase assays using pNPP have been previously described and were performed with the following modifications (Worby et al, 2006;Gentry et al, 2007;Sherwood et al, 2013). Hydrolysis of pNPP was performed in 50 mL reactions, containing 13 phosphatase buffer (0.1 M sodium acetate, 0.05 M bis-Tris, 0.05 M Tris-HCl, pH 7.0, and 2 mM DTT), 50 mM pNPP, and 1 µg of enzyme at 37°C for 15 min.…”