Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity

Saheki, Shûichi; Takeda, Atsushi; Shimazu, Takashi

doi:10.1016/0003-2697(85)90229-5

Cited by 205 publications

(150 citation statements)

References 6 publications

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“…The phosphorylase activity of the enzyme suspension was determined by quantifying the concentration of liberated P i according to Saheki, Takeda, and Shimazu (1985). One enzyme unit (EU) of phosphorylase activity was the amount of enzyme (in ml suspension) releasing 1.0 lmol of P i per minute at 37°C and pH 6.2 (0.10 M sodium citrate buffer).…”

Section: Phosphorylase Activity and Phosphate Determinationmentioning

confidence: 99%

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

Putseys

Derde

Lamberts

et al. 2009

Carbohydrate Polymers

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a b s t r a c tWhen amylose was synthesized using potato phosphorylase in the presence of amylose complexing lipids, monodisperse populations of amylose-lipid complexes were formed. Enzyme dosage and glucose-1-phosphate (glc-1-P)/primer ratio influenced the reaction rate of the enzymic synthesis, presumably by changing the balance between amylose synthesis and amylose-lipid complexation and precipitation, and impacted the molecular weight of the complexes. Lipid characteristics affected the dissociation properties and amylose chain lengths of the amylose-lipid complexes presumably by determining the minimal amylose chain length necessary for complexation and precipitation. Tailor made short chain amylose-lipid complexes can hence be produced by choosing the appropriate reaction conditions. We propose a synthesis mechanism in which the primer is elongated until an amylose chain is obtained which is of sufficient length to complex a first lipid. Further chain extension then occurs, together with subsequent complexation until the complex becomes insoluble and precipitates.

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Section: Phosphorylase Activity and Phosphate Determinationmentioning

confidence: 99%

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

Putseys

Derde

Lamberts

et al. 2009

Carbohydrate Polymers

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“…Assays for M6P isomerase (phosphomannose isomerase) and mannitol 1-P phosphatase have been described (27), or were modified here using a different phosphate assay (28) for the phosphatase activity. …”

Section: Enzyme Assaysmentioning

confidence: 99%

Mannitol Synthesis in Higher Plants

Loescher¹,

Tyson²,

Everard³

et al. 1992

Plant Physiol.

120

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Mannitol is a major photosynthetic product in many algae and higher plants. Photosynthetic pulse and pulse-chase 14C-radiolabeling studies with the mannitol-synthesizing species, celery (Apium graveolens L.) and privet (Ligustrum vulgare L.), showed that mannose 6-phosphate (M6P) and mannitol 1-phosphate were among the early photosynthetic products. A NADPH-dependent M6P reductase was detected in these species (representing two different higher plant families), and the enzyme was purified to apparent homogeneity (68-fold with a 22% yield) and characterized from celery leaf extracts. The celery enzyme had a monomeric molecular mass, estimated from mobilities on sodium dodecyl sulfate-polyacrylamide gels, of 35 kilodaltons. The isoelectric point was pH 4.9; the apparent Km (M6P) was 15.8 millimolar, but the apparent Km (mannitol 1-phosphate) averaged threefold higher; pH optima were 7.5 with M6P/NADPH and 8.5 with mannitol 1-phosphate/NADP as substrates. Substrate and cofactor requirements were quite specific. NADH did not substitute for NADPH, and there was no detectable activity with fructose 6-phosphate, glucose 6-phosphate, fructose 1-phosphate, mannose 1-phosphate, mannose, or mannitol. NAD only partially substituted for NADP. Mg2+, Ca2+, Zn2+, and fructose-2,6-bisphosphate had no apparent effects on the purified enzyme's activity. In vivo radiolabeling results and the enzyme's kinetics, specificity, and distribution (in two-plant families) all suggest that NADPHdependent M6P reductase plays an important role in mannitol biosynthesis in higher plants.Sugar alcohols (acyclic polyols or alditols) are obtained when the aldo or keto group of a sugar is reduced to a hydroxyl. Mannitol, the most frequently occurring sugar alcohol in plants, is particularly abundant in algae and has been detected in at least 70 higher plant families. It is a major carbohydrate in many members of some dicot families, e.g. the Scrophulariaceae, Oleaceae, Rubiaceae, and Apiaceae (2). Until recently, however, little information has been available on mannitol's role in higher plants (16,17 that it is an early photosynthetic product (27, 29) and present in phloem tissue or phloem exudates of celery (family Apiaceae) (9) and species in many other families, e.g. the Oleaceae (30). Other physiological roles have been proposed, including osmoregulation, storage and recycling of reducing power, and service as a compatible solute (16,17), but very little is known of mannitol metabolism in higher plants. A M6PR2 has been reported as being located in the cytosol of mesophyll protoplasts from celery (27). Preliminary labeling data derived from celery and privet were responsible for the initial assays for reductase activity with M6P and mannitol 1-P as substrates.Here we demonstrate the formation of M6P and mannitol 1-P as early photosynthetic products in celery and privet (family Oleaceae). We also report evidence for the role and importance ofM6PR and its characteristics in mannitol biosynthesis in celery. MATERIALS AND METHODS Plant Mate...

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“…One adaption of the method is a change in assay conditions to a mild acid buffer at pH 4 or 5, thus reducing the rate of labile P o hydrolysis (Lowry and Lopez 1946;Saheki, Takeda, and Shimazu 1985). This approach has been used in assays of enzymatically P i -releasing reactions from labile P o substrates in biochemical research (Saheki, Takeda, and Shimazu 1985;Drueckes, Schinzel, and Palm 1995). However, the method appears unsuitable for complex agricultural and environmental samples due to the interference of silicate and other impurities (Lowry and Lopez 1946;Saheki, Takeda, and Shimazu 1985).…”

Section: Introductionmentioning

confidence: 99%

“…This approach has been used in assays of enzymatically P i -releasing reactions from labile P o substrates in biochemical research (Saheki, Takeda, and Shimazu 1985;Drueckes, Schinzel, and Palm 1995). However, the method appears unsuitable for complex agricultural and environmental samples due to the interference of silicate and other impurities (Lowry and Lopez 1946;Saheki, Takeda, and Shimazu 1985). Dick and Tabatabai (1977) reduced errors by complexing the excess molybdate ions to prevent further formation of blue color from P i derived from acid labile P hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

A Modified Molybdenum Blue Method for Orthophosphate Determination Suitable for Investigating Enzymatic Hydrolysis of Organic Phosphates

Honeycutt

2005

Communications in Soil Science and Plant Analysis

162

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In characterizing organic phosphorus (P o ) by phosphatase hydrolysis, the quantity of hydrolyzable P o is represented by the difference in orthophosphate [i.e., inorganic P (P i )] determined after and prior to enzymatic incubation. Therefore, precise determination of P i is of major importance for accurate application of the enzymatic hydrolysis approach. The strong acid conditions required for conventional molybdenum blue methods interferes with P i determination due to rapid hydrolysis of labile P o and precipitation of enzymes (proteins). The molybdenum blue method of Dick and Tabatabai in 1977 reduced errors pertaining to nonenzymatic hydrolysis of P o . This study revisited the method, finding that the absorption coefficient at 850 nm was 45 -49% higher than at 700 nm, and linear up to at least 80 nmol P i in 1-mL assay solution. Therefore, adaptation of the readings at 850 nm improved the sensitivities of P i determination by about 45%. Enzyme precipitation during P i determination was prevented by addition of 2% sodium dodecyl sulfate (SDS) before color-forming reagents were added. This method modification provides increased sensitivity for P i determination, thereby improving the accuracy of P o analysis by phosphatase hydrolysis.

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Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity

Cited by 205 publications

References 6 publications

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

Production of tailor made short chain amylose–lipid complexes using varying reaction conditions

Mannitol Synthesis in Higher Plants

A Modified Molybdenum Blue Method for Orthophosphate Determination Suitable for Investigating Enzymatic Hydrolysis of Organic Phosphates

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