Exopolyphosphatase Activity Research Articles

Purification and characterization of an exopolyphosphatase from Saccharomyces cerevisiae.

An exopolyphosphatase (polyphosphate phosphohydrolase; EC 3.6.1.11) activity that cleaves inorganic polyphosphates to orthophosphate has been purified to apparent homogeneity (> 95% pure) from Saccharomyces cerevisiae. The exopolyphosphatase is a monomeric protein with a polypeptide molecular mass of 28 kDa. The enzyme, which can be stabilized in the presence of Triton X-100, has a pH optimum of 7.5 and requires, for maximal activity, Co2+ or Mg2+ ions. In the absence of these ions, the exopolyphosphatase binds to polyphosphate but does not degrade it, allowing affinity purification of the enzyme on a polyphosphate-modified zirconia support. o-Vanadate, Cu2+, and Ca2+ are effective inhibitors of the exopolyphosphatase. The enzyme preferentially hydrolyzes linear polyphosphates in a non-processive manner; pyrophosphate as well as cyclic tri- and tetrametaphosphate are degraded only at very low rates, whereas ATP is not split by the exopolyphosphatase. The only product formed by the action of the enzyme is orthophosphate.

Determination of Endopolyphosphatase Using Polyphosphate Glucokinase

A method has been developed for determining endopolyphosphatase (polyphosphate depolymerase, EC 3.6.1.10) activity. The enzyme catalyzes the hydrolysis of inorganic polyphosphates [poly(Ps)] by cleaving internal phosphoanhydride bonds without removal of terminal phosphate residues. During the reaction, shorter poly(P) chains are formed and the molar concentration of poly(P) increases. This enzymatic activity is difficult to quantitate, because the substrates and products of the reaction are chemically identical. The commonly used viscometric method lacks sensitivity and cannot be used with shorter poly(P) substrates. The method described here overcomes these problems, and in addition is rapid, simple, and can be used for distinguishing between the endopolyphosphatase and exopolyphosphatase (EC 3.6.1.11) activities. It is based on monitoring the increase in the number of poly(P) chains generated by endopolyphosphatase. For this purpose, the method takes advantage of the specific property of poly(P) glucokinase (EC 2.7.1.63) which utilizes poly(Ps) of different sizes present in the endopolyphosphatase reaction mixture and reduces them to fairly uniform very short-chain product, poly(P)m. The concentration of poly(P)m is expressed in terms of acid-labile phosphorus and is proportional to the duration of the endopolyphosphatase reaction (i.e., the number of original poly(P) chains) and to protein concentration. The increase in the poly(P)m concentration is a relative measure of the endopolyphosphatase activity. Under certain conditions, m equals 3.5 and the activity can be expressed in standard units, since the exact number of poly(P) chains formed by endopolyphosphatase can be calculated from the increase in molar concentration of poly(P)3.5. Accuracy and advantages of the assay are discussed.

Phosphorus assimilation i mycorrhizal moong ( Vigna radiata L.) plants under different phosphorus levels

Influence of phosphorus on its assimilation was studied in a pot experiment at two growth stages at different phosphorous (P) levels control (P 0), 10 μg P g −1 soil (P 10) and 20 μg P g −1 soil (P 20) in non-mycorrhizal (NM) and mycorrhizal (M) moong plants. The response of M plants to P application varied both with growth stage and P level. In NM plants, total P, shoot fresh weight (FW) and exopoly-phosphatase (ExoPase) activity in roots was enhanced with both P levels while root peroxidase and acid phosphatase activities (APase) in leaves increased with P 20 level only. Ester P, root FW, inorganic phosphate (P i) and APase activity in roots were not affected at P 10 level. Total P and P i in leaves were enhanced with MP 10 but were unaltered with MP 20 treatment at 38 days after sowing (DAS). ExoPase and peroxidase activities increased but total P in leaves decreased with MP 10 and MP 20 plants at 50 DAS. APase activity and ester P in leaves increased with MP 20 while Chl concentration decreased at 38 as well as 50 DAS. Soil available P increased with both levels in M plants but only with P 20 with NM plants. The comparison of M and NM plants showed that M plants had greater P i at both samplings whereas root total P and Chl only at 50 DAS at each P level. Shoot FW and root ExoPase activity were greater in M plants with P 0 and P 10 levels. NM plants accumulated ester P more in roots while M plants in leaves at 50 DAS.

THE ENZYMES OF POLYPHOSPHATE METABOLISM IN VESICULAR‐ARBUSCULAR MYCORRHIZAS

SUMMARYThe presence and distribution of enzymes of polyphosphate (polyP) synthesis and degrActation was examined in mycorrhizal and non‐mycorrhizal onion roots, and both external and internal hyphae of the endophyte, Glomus mosseae. PolyP kinase activity was detected in extracts of external hyphae and mycorrhizal roots. Activity was dependent on exogenous polyP and Mg2+, the reaction product being a long chain polyP. Exopolyphosphatase activity was detected in both mycorrhizal and non‐mycorrhizal roots but activity was 134°O greater in the former. Endopolyphosphatase activity was also greater in mycorrhizal roots. Polyphosphatases were detected in extracts of internal hyphae, but not external hyphae. Polyphosphate glucokinase activity was demonstrated in extracts of external hyphae and mycorrhizal roots, the immediate reaction product being glucose‐6‐phosphate. These results are discussed in relation to potential mechanisms of P translocation and transfer in vesicular‐arbuscular (VA) mycorrhizas. A novel technique for isolating the internal hyphae and associated vesicles and arbuscules from mycorrhizal onion roots is described.