An Alternative Method for Enzymatic Assay of Plant Invertases

Abstract

An alternative coupled method was developed for the enzymatic assay of plant invertases. In this method, ADP produced from the phosphorylation of glucose and fructose (hydrolysis products of invertases) is coupled to oxidation of NADH by the enzymes pyruvate kinase and lactate dehydrogenase in the presence of phosphoenolpyruvate and NADH. This method was compared with the glucose-6-phosphate dehydrogenase method for both continuous and discontinuous assay by using purified invertase from baker's yeast and protein preparations derived from plant materials of three different species. This method is applicable to filtered and concentrated crude extracts. Statistical analysis indicated that the alternative method was similar in accuracy to the glucose-6-phosphate dehydrogenase method. Keywords: Biochemistry; enzymes; physiology; sugars; invertase

Changes in Invertase Activities and Reducing Sugar Content in Sweetpotato Stored at Different Temperatures

“…Neutral (pH 7.0) and alkaline (pH 7.6) invertase activities were determined by continuous assay (Huang et al, 1998) Acid invertase assay followed the procedure of Robyt and White (1987) with a slight modification. Ten microliters of enzyme concentrate was added to 990 µL of 50 mM incubation buffer (25 mM citrate/25 mM ADA, pH 4.6) and incubated for 1 min.…”

“…Each extraction consisted of a composite sample of 10 g of internal flesh tissue from four different roots. Crude extracts were prepared as described (Huang et al, 1998). The resultant crude extracts were vacuum-filtered five times through two layers of Whatman No.…”

“…Invertase Assays. Neutral (pH 7.0) and alkaline (pH 7.6) invertase activities were determined by continuous assay (Huang et al, 1998) in 50 mM N-(2-acetamido)iminodiacetic acid (ADA; pH 7.0) and 50 mM (HEPES)-KOH (pH 7.6), respectively. Each buffer contained 10 mM MgCl2 and 20 mM KCl to meet the specific requirements of the auxiliary enzymes * Author to whom correspondence should be addressed [telephone (225) 388-3862; fax (225) for metal ions.…”

Proteomic insight into soybean response to flooding stress reveals changes in energy metabolism and cell wall modifications

“…Plant invertase assay was performed by slightly modifying protocol of Huang et al [ 31 ]. The extraction procedure was performed on ice.…”

A Continuous Method for Enzymatic Assay of Sucrose Synthase in the Synthetic Direction

“…The observations at the linear part of the curve were used to calculate the slope, which is the rate of product formed versus time. SS activity was determined by regression from the initial slope of the curve (Huang et al, 1998).…”