Enzyme-Linked Immunosorbent Assay of Fungal NADP⁺-Glutamate Dehydrogenase

Abstract

A sensitive and reliable method has been developed for the quantitation of NADP+-glutamate dehydrogenase from the phytopathogenic Ascomycete Splhaerostilbe repens using a two-step competitive enzyme-linked himunosorbent assay. Purified enzyme was adsorbed noncovalently to polystyrene wells and rabbit immunserum was allowed to bind to antigensensitized wells. Bound specific antibody was visualized by goat antirabbit immunoglobulin covalently linked to alkaline phosphatase using paranitrophenylphosphate as the substrate. Increasing amounts of purified enzyme or crude fungal extracts were quantitated by their ability to inhibit specific antibody adsorption to antigen-coated polystyrene wells. This system proves to be useful in the range of 10 to 80 nanograms of enzyme level.Using this assay, identical amounts of NADP'-glutamate dehydrogenase were found in mycelia grown on nitrate and ammonia sources.A primary research goal in our laboratories is to elucidate the biochemical mechanisms which regulate the enzymes of nitrogen metabolism levels in parasitic and symbiotic fungi under different nutritional conditions and during the cell cycle (8,9 Culture Conditions and Harvest Procedures. Sphaerostilbe repens (strain CBS 275-60) was supplied by the Centraalbureau voor Schimmelcultures of Baarn, the Netherlands. The mycelium was grown in liquid culture and harvested as described previously (2).Enzyme Activity and Total Protein Assay. A spectrophotometric assay (8) was used to measure the amination activity ofthe NADP-GDH. Enzyme activity was expressed in nkat. The total protein concentration was determined according to Lowry et al. (7).Purification of NADP-GDH. All purification steps were carried out at 4°C. Buffers were adjusted to the indicated pH at 20°C. The NADP-GDH of S. repens was purified by chromatography on hydroxylapatite (Biogel HTP), DEAE-Sephacel, and preparative polyacrylamide gel electrophoresis (see below).Approximately 720 g fresh weight of frozen mycelia were thawed, ground with acid-washed sand in a mortar, in 2.5 L 0.1 M K-phosphate (pH 7.5) containing 14 mm 2-mercaptoethanol and 70 g insoluble PVP (Polyclar AT). The resulting homogenate was filtered through two layers of cheesecloth and centrifuged for 20 min at 40,000g. The supernatant was brought to 55% (NH4)2SO4 saturation, centrifuged, and the supernatant was adjusted to 70%o saturation with this salt. The protein precipitate was recovered by centrifugation and dissolved in 0.01 M K-phosphate (pH 7.0). This solution was applied to a column (3 x 30 cm) of Sephadex G-25 equilibrated and eluted with K-phosphate (pH 7.0). The desalted proteins were collected and were layered on the top of a column (2.5 x 20 cm) of Biogel HTP equilibrated with the same buffer.The column was washed with the buffer, and the enzyme was then eluted with a 300-ml linear gradient from 0.01 to 0.3 M K-398 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Spectrofluorometric Determination of Trace Amounts of Coenzyme II Using Norfloxacin-Terbium Complex as a Fluorescent Probe

“…The familiar methods for the determination of NADP include electrochemical methods, [1][2][3][4][5][6][7][8] HPLC, 9-17 microfluorometric method, 18 bioluminescence method, 19 spectrophotometric method, 20 enzyme-linked immunosorbent assay, 21 capillary electrophoresis method 22 and FIA-CME. 23,24 But there was no report about a spectrofluorometric method for determination of NADP using Tb 3+ -NFLX as fluorescent probe.…”

Spectrofluorimetric determination of trace amount of coenzyme II using ciprofloxacin–terbium complex as a fluorescent probe

“…Therefore, it is significant to establish a precise, sensitive and simple method for the determination of NADP. Now the familiar methods for the determination of NADP include electrochemical methods [1][2][3][4][5][6][7][8], HPLC [9][10][11][12][13][14][15][16][17], microfluorimetric method [18], bioluminescence method [19], spectrophotometric method [20], enzyme-linked immunosorbent assay [21], capillary electrophoresis method [22], FIA-CME [23,24], etc. But there was no report about spectrofluorimetric method for determination of NADP using Tb 3+ -CIP as fluorescent probe.…”