Abstract
In phosphorylase assays in crude yeast extracts with glucose-1-phosphate (G-1-P) as substrate, 25–30% of the Pi-liberating activity could not be inhibited by antibodies against yeast phosphorylase and were attributed to the action of phosphatases. During phosphorylase preparation from baker's yeast (Saccharomyces cerevisiae), a phosphatase, molecular weight 45000±5000, with high specificity for G-1-P, pH-optimum 5.6, was isolated which appeared to be responsible for the interference. It did not hydrolyze other glycolytic intermediates, pyrophosphate or adenylates. No activation by Mg2+ or inhibition by (+)-tartrate, and only 40% inhibition by 50 mM F- were observed, 5,5′ dithiobis-(nitrobenzoic acid) (10mM) inactivated the enzyme completely. Its affinity for G-1-P was very low (K m=40 mM). Consequently, it mainly interfered with the phosphorylase assay in the amylose synthesizing reaction, in which high G-1-P-concentrations have to be used. For phosphorylase assays in crude extracts, measurement of the phosphorolytic activity is recommended, in which the concentration of G-1-P is kept sufficiently low.
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Abbreviations
- G-1-P:
-
Glucose-1-phosphate
- (NbS)2 :
-
5,5′ dithiobis-(2-nitrobenzoic acid)
- SDS:
-
Sodium dodecylsulfate
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Becker, JU. Isolation and characterisation of an acid phosphatase interfering with phosphorylase determinations in crude extracts from yeast. Arch. Microbiol. 123, 233–238 (1979). https://doi.org/10.1007/BF00406655
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DOI: https://doi.org/10.1007/BF00406655