Enzyme characterization and assay procedures

1. Nitrate reductase

Nitrate reductase (NR) is a labile enzyme and for this reason a number of protectants are added to the extraction media (Wray and Fido, 1990). These protectants usually include nitrate, which is an NR substrate; FAD, which is a prosthetic group present in the protease-sensitive region of NR; EDTA, which chelates toxic metals released during cell breakage; and sulphydryl compounds such as dithiothreitol and mercap-toethanol, which prevent the oxidation of essential SH-groups of NR. Protease inhibitors such as phenylmethylsulphonyl fluoride are usually added to inhibit proteolysis during extraction. Exogenous proteins such as bovine serum albumin and casein are routinely used to enhance the stability of NR. A detailed study of in vitro measurement of NR in the basidiomycete Hebeloma cylindrosporum has been carried out by Plassard et al. (1984a).

Nitrate reductase can be assayed directly in crude cell-free preparations. The overall physiological reaction of NR is usually determined by the reduction of nitrate in the presence of NAD(P)H followed by the colorimetric measurement of the nitrite produced:

In higher plants, nitrate reductase is NADH-specific, whereas in fungi the enzyme shows a preference for NADPH (Beevers and Hageman, 1980). In Hebeloma cylindrosporum the activity of NR is shown to be strictly dependent on NADPH as the electron donor (Plassard et al., 1984a).

The following assay procedure is a modification of Scholl et al. (1974). The reaction mixture contains 25 mM potassium phosphate (pH 75), 10 mM potassium nitrate, 0.5 mM NADH or NADPH and up to 0.25 ml enzyme in a final volume of 0.5 ml. The reaction is carried out for 20-30 min at 25 °C and stopped by adding 0.25 ml of a 200 mM zinc acetate solution. The precipitated material is cleared by centrifugation. The residual pyridine nucleotide in the reaction mixture is oxidized by adding 0.25 ml of a freshly prepared 50 ¿iM phenazine methosulphate solution and leaving the mixture at room temperature for about 20 min. The colour development is initiated by adding 1 ml each of the diazo-coupling reagents 1% sulphanilamide solution in 3 n HC1 and 0.02%

yV-(l-naphthyl)-ethylenediamine dihydrochloride solution in 0.1 n HC1. After 20 min the absorbance of the pink diazo dye is read at 540 nm and the amount of nitrite is determine from a standard curve for 10-100 nmol nitrite.

The Km for nitrate and NADPH of the fungal nitrate reductase range from 60-200 /xM and 9-60 /u,M, respectively (Beevers and Hageman, 1980). The Km values for nitrate and NADH are similarly low for the plant enzyme. Nitrate reductase is a substrate-induced enzyme and is usually found only in the presence of nitrate. In fungi, the presence of ammonium in the culture medium completely abolishes the nitrate-mediated induction of nitrate reductase (Lewis and Fincham, 1970). A similar inhibition of nitrate reductase induction has been shown in plant roots (Smith and Thompson, 1971; Radin, 1975).

2. Nitrite reductase

One of the major differences between the plant and fungal nitrite reductase (NiR) is in their specificity for electron donors. The plant enzyme accepts electrons from reduced ferredoxin whereas the fungal enzyme utilizes pyridine nucleotides as the electron donor and generally shows a preference for NADPH. The reaction catalysed by NADPH-NR is considered to involve a sequential transfer of electrons:

NADPH-NR extracted from Hebeloma cylindrosporum shows a rapid loss of activity but can be reactivated by the addition of sodium dithionite and methylviologen as the electron donor (Plassard et al., 1984b). These authors have suggested that the loss of NADPH-NR activity in the fungal extract was associated with the step involved in the transfer of electrons from NADPH to FAD, and the reduction of siroheme by methylviologen bypassed the need for electrons from NADPH. The overall reaction of NiR can be summarized as:

Nitrite + reduced ferredoxin or NAD(P)H

NH4 + oxidized ferredoxin or NAD(P)

The activity of nitrite reductase is usually measured by determining the disappearance of nitrite in the assay system. For pyridine nucleotide-dependent assay, the following modification of Garret (1972) may be used. The reaction mixture contains 50 mM potassium phosphate (pH 7.5), 1 mM potassium nitrite, 0.05 mM FAD, 0.5 mM NADPH or NADH and enzyme in a final volume of 0.5 ml. After 15 or 20 min incubation at 25 °C, the reaction is stopped and the amount of nitrite in the sample is determined according to the procedure described for nitrate reductase assay. For assaying ferredoxin-dependent nitrite reductase activity, the above reaction mixture is modified to substitute NAD(P)H by 0.5 mM ferredoxin and 12 mM sodium dithionite. The reaction is started by the addition of dithionite and stopped by vigorous shaking. Residual nitrite is measured as above.

As with nitrate reductase, nitrite reductase is a high affinity enzyme and shows Km values for nitrite in the micromolar range. The fungal and plant NiR differ considerably in their molecular size. In higher plants NiR has a molecular mass of about 63 kDa (Wray and Fido, 1990) which is four times lower than the molecular weight of 290 kDa reported for NiR isolated from Neurospora crassa (Nason et al., 1954). The molecular mass of the purified NiR is usually determined by gel chromatography.

3. Glutamate dehydrogenase

The in vitro activity of glutamate dehydrogenase (GDH) enzymes can be assayed both in the aminating (assimilatory) and deaminating (cata-bolic) directions.

The root tissue of higher plants usually contains a mitochondrial NADH-linked GDH, which functions primarily in the deaminating direction. Many fungi possess both the NADH-linked catabolic enzyme and an NADPH-linked assimilatory GDH which can be separated in a one step anion-exchange chromatographic procedure (Fig. 2).

The usual assay system of GDH activities is based on spectrophoto-metric monitoring of the oxidation of NAD(P)H, which gives a stoichiometric measure of the amount of glutamate produced. The following is a modification of Ahmad and Hellebust (1984). The reaction mixture of 1 ml contains (final concentration) 50 mM potassium phosphate (pH 7.5), 100 mM ammonium chloride, 20 mM 2-oxoglutarate, 0.2 mM NADH or NADPH and 0.2 ml enzyme. The oxidation of NAD(P)H is monitored at 340 nm using an extinction coefficient (e) of 6.2. The assay must be corrected for non-specific oxidase activities present in the enzyme extract. These contaminating activities can be determined by omitting 2-oxoglutarate from the reaction mixture. Membrane-bound oxidases can be removed either by 20% ammonium sulphate precipitation followed by centrifugation at 10000 g for lOmin (Ahmad and Hellebust, 1986) or by high speed (30 000 g) centrifugation for 30 min (Ahmad et al., 1990).

3.. glutamate dehydrogenasi

3.. glutamate dehydrogenasi

100-

glut amine synthetase glut amine synthetase

j.. alanine aminotransferase j.. alanine aminotransferase

Fraction number

Fig. 2. Elution profiles for the fractionation by anion-exchange chromatography of glutamate dehydrogenase (NADH-GDH, closed symbols; NADPH-GDH open symbols); glutamine synthetase (transferase activity, closed symbols; synthetase activity, open symbols) and alanine aminotransferase. A 4 ml extract prepared by grinding 1 ml packed volume of Laccaria bicolor was clarified by centrifugation (30000 g, 30 min) and a 2 ml fraction loaded onto a Mono-Q column attached to a fast protein liquid chromatography assembly. The composition of the elution media was as described by Ahmad and Hellebust (1987).

The pH optima of the amination reactions of both NADH-GDH and NADPH-GDH from various sources fall in the pH range 7-8, whereas the pH optima of the deaminating reactions are usually 1.0-1.5 pH units higher. These differences in pH optima appear to have broad implications for the functional relations of these enzymes. NADH-GDH from various sources have a high Km for ammonium (10-80 mM). The Km for ammonium of NADPH-GDH from higher plants and many fungi is also high. However, NADPH-GDH from some ectomycorrhizal fungi show biphasic kinetics with different Km values for ammonium (Martin et al., 1983; Ahmad et al., 1990). The lower Km value for these NADPH-GDH enzymes is in the range of 2-5 mM ammonium. The

NADH-linked GDH of higher plants is a metalloprotein and requires added calcium to prevent inactivation by EDTA or other chelating agents. No calcium requirement has been reported for the fungal GDH enzymes.

4. Glutamine synthetase

Two separate forms of glutamine synthetase (GS) exist in higher plants; one located in the cell cytosol and the other in plastids. GS activity in plant roots is shown to be predominantly cytosolic (Emes and Fowler 1979; Suzuki et al., 1981). The anion-exchange study of GS in the ectomycorrhizal fungus, Laccaria bicolor, shows the presence of a single molecular form (Fig. 2) with an elution profile on a sodium chloride gradient similar to that observed for cytosolic enzymes from many plants and algal sources (McNally et al., 1983; Casselton et al., 1986). The plastid enzyme requires the presence of sulphydryl reagents to prevent inactivation during isolation. However, these reagents are shown to suppress the activity of cytosolic GS from some sources (Wallsgrove et al., 1983; Ahmad and Hellebust, 1987). It may therefore be necessary to isolate the two GS forms by separate extraction procedures with appropriate sulphydryl composition of the isolation media. The two GS isoforms are also reported to differ in their thermal stability with the cytosolic form being considerably less labile (Mann et al., 1979; Ahmad et al., 1982).

GS catalyses the formation of glutamine from ammonium and glutamate at the expense of ATP hydrolysis; the overall reaction can be summarized as:

This reaction can be determined by a coupled spectrophotometric assay procedure where the ADP produced in GS reaction is linked to the conversion of phosphoenolpyruvate by pyruvate kinase to pyruvate, which in turn is linked to the oxidation of NADH by lactate dehydrogenase. The following is a modification of Stewart and Rhodes (1977a). The reaction mixture of 1 ml is prepared with 50 mM sodium phosphate buffer (pH 7.5) containing 100 mM sodium glutamate, 10 mM ATP, 10 mM ammonium chloride, 20 mM magnesium sulphate, 1 mM phosphoenolpyruvate, 0.2 mM NADH, 5 units pyruvate kinase (PK), 2 units lactate dehydrogenase (LDH) and 200 yu.1 enzyme extract. The oxidation of NADH monitored at 340 nm gives a stoichiometric estimate of the amount of glutamine produced by the GS reaction.

A colorimetric procedure has been developed by substituting hydroxylamine (NH2OH) for ammonium and determining the amount of y-glutamylhydroxamate produced spectrophotometrically following the development of a brown colour with ferric chloride.

Glutamate + NH2OH + ATP

y-Glutamylhydroxamate + ADP + Pi + H20

This alternative assay gives a measure of GS reaction in the biosynthetic direction and is usually termed the synthetase assay. The Vmax and values determined by this method are usually similar to those obtained by a coupled assay system. The reaction is started in a 1 ml mixture prepared with 100 mM Tris-HCl buffer (pH 7.6) and containing 80 mM glutamate, 20 mM hydroxylamine, 20 mM ATP, 50 mM magnesium sulphate and 200 fil enzyme extract (Ahmad and Hellebust, 1984). After a desired period of incubation (20-30 min) at 25 °C, the reaction is stopped by the addition of 1 ml of acidified ferric chloride solution (26 g of ferric chloride and 40 g of TCA in one litre of 1 n HC1). It is essential to run zero time controls by stopping the reaction immediately after the addition of enzyme extract to the reaction mixture. Where necessary, the precipitated material is removed by sedimentation in a bench-top centrifuge before reading the aborbance at 540 nm. A standard plot using a commercial glutamylhydroxamate preparation is established as a reference for GS activity.

A third assay has been developed on the basis of the ability of GS to catalyze the y-glutamyl transfer reaction that results in the formation of y-glutamylhydroxamate from glutamine and hydroxylamine:

Glutamine + NH2OH y-Glutamylhydroxamate + NH|

This reaction—termed transferase assay—gives rates several times higher than those obtained by the above biosynthetic reactions. After establishing the transferase synthetase ratio of a given GS enzyme, this method can be used as a sensitive indicator of its activities at different stages of purification protocols. The transferase assay is based on the colorimetric determination of glutamylhydroxamate produced. The reaction is started in a 1 ml mixture with 100 mM Tris-acetate buffer (pH 6.4) containing 100 mM glutamine, 30 mM hydroxylamine, 30 mM sodium arsenate, 1.5 mM MnCl2, 0.2 mM ADP and 200 (¿1 enzyme (Ahmad and Hellebust, 1984). The reaction is stopped after 30 min by the addition of acidified ferric chloride and GS activity is quantified as described for the synthetase assay.

The kinetics of GS have been extensively studied. Various purified preparations have been shown to have high affinity for ammonium, but a considerably lower affinity for glutamate (Stewart et al., 1980). The ATP-dependent reaction of glutamine synthetase has a specific requirement for magnesium. The enzyme shows complex kinetics with respect to concentrations of ATP and magnesium and pH of the reaction system (Stewart etal., 1980).

5. Glutamate synthase

Glutamine produced via the glutamine synthetase pathway is utilized by glutamate synthase (GOGAT), which catalyses the transfer of amido nitrogen to 2-oxoglutarate, resulting in the formation of two molecules of glutamate. The enzyme from green plants is specific for ferredoxin or ferredoxin-like proteins (Miflin and Lea, 1980; Suzuki et al., 1985) as the electron donor

Glutamine + 2-oxoglutarate + reduced ferredoxin C^4T

2 Glutamate + oxidized ferredoxin whereas GOGAT from various non-green micro-organisms is found to be NAD(P)H-specific (Stewart et al., 1980):

2 Glutamate + NAD(P)

The net result of the combined action of GS and GOGAT is the synthesis of glutamate from ammonium and 2-oxoglutarate; this combined action is frequently referred to as the GS-GOGAT cycle or simply as the glutamate synthase pathway.

GOGAT can be measured directly in crude extracts. The enzyme is usually extracted in 50 raM phosphate buffer (pH 7.5) containing protectants such as sulphydryl reagents, mercaptoethanol and DTT, substrate 2-oxoglutarate and protease inhibitor PMSF (Marquez et al., 1988; Wallsgrove et al., 1977). In some cases it is necessary to solubilize membrane-associated ferredoxin-dependent GOGAT from plant tissue by the addition of 0.05% to 0.5% Triton X-100 to the extraction media. GOGAT activity is usually measured by the quantitative measurement of glutamate produced. For assaying in vitro ferredoxin-dependent GOGAT activity the electron donor can be replaced by reduced methyl viologen without significant change in activity (Marquez et al., 1988). The enzyme extract is pre-incubated for 20 min in a 0.5 ml mixture containing 100 mM phosphate buffer, pH 7.5, 10 mM glutamine, 10 mM 2-oxoglutarate and 15 mM methyl viologen. The reaction is started by adding 100 ¡¿1 of freshly prepared dithionite reductant mixture (235 mg sodium dithionite, 250 mg sodium bicarbonate in 5 ml water). In NAD(P)H-dependent GOGAT assays, methyl viologen in the reaction mixture and its reduction by dithionite mixture are omitted, and the reaction is started by the addition of 0.5 mM NADH or NADPH. After

20 min at 25 °C, the reaction is stopped either by boiling or by the addition of 1 ml ethanol. Glutamate produced can be separated from glutamine and quantified by one of the chromatographic procedures employing paper chromatography (Wallsgrove et al., 1977), thin layer chromatography (see Lea et al., 1990), high-performance liquid chromatography (Martin et al., 1982), anion-exchange column chromatography (Hecht et al., 1988) or paper electrophoresis (Chen and Cullimore, 1988). Ninhydrin-based colorimetric determination of glutamate is routinely applied in these studies. The use of radiolabeled 14C glutamine in the reaction mixture allows a more sensitive determination of glutamate by scintillation counting (Wallsgrove et al., 1982). Pyridine nucleotide-dependent GOGAT activity can be measured spectrophotometrically by following the oxidation of NAD(P)H provided the enzyme preparation does not contain non-specific NAD(P)H oxidase activities. The composition of the reaction mixture for spectrophotometric measurements is the same as that described above for NAD(P)H GOGAT activity except for the concentration of NAD(P)H, which is lowered to 0.25 mM to give an initial absorbance reading of less than 2.

6. Aminotransferases

Glutamate synthesized either via the GDH pathway or the GS-GOGAT cycle is the primary amino donor for the synthesis of most other amino acids. Several enzymes catalysing the transfer of amino groups from glutamate to different keto acids have been identified in plants and fungi. The most active of these aminotransferases are aspartate aminotransferase (AsAT) and alanine aminotransferase (A1AT). The reactions of both aminotransferases are reversible:

AsAT

Glutamate + oxaloacetate ^ Aspartate -I- 2-oxoglutarate

Glutamate + pyruvate ^ Alanine + 2-oxoglutarate These enzymes are routinely measured spectrophotometrically by coupling the production of keto acids to the oxidation of NAD(P)H catalyzed by an auxiliary dehydrogenase enzyme. A typical reaction mixture for AsAT assay contains in a final volume of 1 ml, 50 mM sodium phosphate buffer (pH 7.5), 100 mM sodium aspartate, 10 mM 2-oxoglutarate, 0.25 mM NADH, 2 units malate dehydrogenase and purified enzyme extract (Ahmad and Hellebust, 1989).

The reaction mixture for A1AT assay is similar in its composition to the reaction mixture for AsAT except for aspartate and for malate dehydrogenase, which are replaced by 100 mM alanine and 2 units of lactate dehydrogenase, respectively. When the coupled reaction proceeds linearly, the oxidation of NADH gives a stoichiometric measure of aminotransferase activity. The spectrophotometric procedure for measuring aminotransferase activities using glutamate as the amino donor has been described elsewhere (Ahmad and Hellebust, 1989). Enzyme preparations clarified by high speed centrifugation (30000 g, 30 min) and/or ammonium sulphate precipitation are usually adequate for the coupled enzyme assays. The reactions catalysed by aminotransferases require pyridoxal 5'-phosphate as a coenzyme, but in plants and fungi it usually remains tightly bound to the enzyme during different stages of purification and therefore its addition to the reaction mixture is generally not needed. Several isoforms of both alanine and aspartate aminotransferases have been identified from various plant and microbial sources. Figure 2 shows the elution profile by anion-exchange chromatography of alanine aminotransferase from the ectomycorrhizal fungus, Laccaria bicolor. The fungal extract is fractionated into two peaks of A1AT activity; eluting first a cationic minor isoform followed by a major anionic isoform. Cationic aminotransferases are considered mitochondrial in origin whereas anionic isoforms are thought to be cytosolic enzymes (Givan, 1980).

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