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Hirasawa, E. & Suzuki, Y. (1975) Phytochemistry 14,99-101 McGowan, R. E. & Muir, R. M. (1971) Plant Physiol. 47, 644-648 Smith, T. A. (1974) Phytochemiktry 13,1075-1081 Smith, T. A. (1975) Phytochemistry 14,865-890 Smith, T. A. & Bickley, D. A. (1974) Phytochemistry 13, 2437-2443 Tabor, C. W. & Kellogg, P. D. (1970) J. Biol. Chem. 245, 54245433 Weaver, R.H. & Herbst, E. J. (1958) J. Biol. Chem. 231,647-655 Yamada, H., Tanaka, A. & Ogata, K. (1965) Agric. Bid. Chem. 29, 260-261 Yamasaki, E. F.,Swindell, R. & Reed,D. J. (1970) Biochemistry 9, 1206-1210

Reconstitution in vitro of Nitrate Reductase from Apoprotein of Molybdenum-Deficient Spinach GARRY RUCKLIDGE, BRIAN NOlTON and ERIC HEWIlT

Long Ashton Research Station, Universityof Bristol, Long Ashton, BristolBSl8 9AF, U.K. Reconstitution in vitro of fungal NADPH-nitrate reductase by complementation of mutant proteins or by using acid-treated molybdoproteins to produce a molybdenum coordinating component to combine with a mutant protein product has been reviewed by Hewitt (1975). We now report formation of NADPH-nitrate reductase in uitro in higher-plant preparations of molybdenum component and apoprotein lacking molybdenum. Spinach plants were grown in sand culture with a complete nitrate nutrient (Hewitt, 1966). After 5 weeks leaves (50-100g) were macerated with 2.5ml of 0.1 wNaH2PO4/ Na2HP04/1mM-EDTA extracting buffer (pH7.5)/g and centrifuged at 59000g.,. for 30min. Nitrate reductase in the supernatant was twice precipitated with (NH4),S04 at 50% saturation. The precipitate was dissolved in extracting buffer and a 2nd sample applied to a Sephadex G-100 column (1.5cm x 32cm) equilibrated with the same buffer. Fractions having nitrate reductase activity were pooled and stored at 4 ° C .Apoprotein was obtained by macerating (in 2ml of buffer) 1g of leaves from plants grown with a molybdenum-deficient nutrient containing additional (NH.&sO4 ( 4 m ~(Notton ) et al., 1974). The macerate was centrifuged as above and the supernatant used immediately. All above operations were at 04°C. Sucrose-density centrifugation was performed on an MSE Superspeed 75 and with a 3 x 6.5ml swing-out rotor at 16OOOOg (ruv.7.1 cm)for 16h at 2°C through 5ml of a 5-20% sucrose gradient containing 1 0 ~ ~ FAD with a 50% sucrose cushion, or 20ml of the same gradient but with a 3x25ml aluminium swing-out rotor at 9OOOOg (r.,. 9.4cm) for 36h where appropriate. Molecular-sieve chromatography of 12ml of apoprotein preparations utilized a Bio-Gel A 0.5m column (2.5cm x 1OOcm) equilibrated with extracting buffer containing 0.1 M-KCI.Nitrate reductase and cytochrome c reductase activities were determined as described by Wray & Filner (1970). Optimum conditions for reconstituting nitrate reductase involved gradual acidification of nitrate reductase preparations to pH2.5 with 1M-HCIfollowed after 3-5min by restoring the solution to pH7.0, separatingthe resultant precipitate by centrifugation and resuspending in 0.1 M-sodium phosphate buffer, pH6.2. This suspension, providing an excess of the molybdenum component, was mixed with an equal volume of apoprotein preparation and preincubated for 4Omin at 20°C before commencement of the enzyme assay. Minimal initial loss and maximal stability of the component depended on the pH of restoration (Fig. 1). Half-lives at 4°C after the initial rapid loss were 69min at pH7.0 and 15min at pH2.5. After reconstitution, nitrate reductase was submitted to sucrose-density centrifugation in 5ml tubes. Fractions were assayed for cytochrome c reductase activity and compared with untreated apoprotein as control. Acid-treated nitrate reductase had neither residual cytochrome c reductase nor nitrate reductase activities. Results (Fig. 2) show that after reconstitution there was an increase in the heavy (8 S) cytochrome c reductase area (Wray & Filner, 1970), where nitrate reductase appeared and is normally Vol. 4

BIOCHEMICAL SOCIETY TRANSACTIONS

\-

Storage time (min)

Storage time (rnin)

Fig. 1. Eflect of p H on time-course of inactivation of molybdenum component during storage at 4°C

A nitrate reductase preparation was acidified at pH2.5 and either left unchanged (A) or readjusted back to pH6.0 (A) or 7.0 (0) or 8.0 (0).The pH2.5 solution or centrifuged precipitates resulting from readjustment of pH were stored at 4°C for periods of time up to 120min before restoration of pH to 6.2 and reconstitution of nitrate reductase with apoprotein as described in the text. The insert shows log (% reconstituted activity) plotted against time.

found, and a corresponding decrease in the lighter (4s) cytochrome c reductase area. Although the lighter protein appeared to provide the reactive species, it was possible that the heavy protein also reacted or was stabilized after reconstitution relative to the lighter protein. Proteins of the light and the heavy regions were separated preparatively either on Bio-Gel A 0.5m or centrifugally in sucrose in the larger rotor. Fractions were selected from just below the lighter peak and just beyond the heavier peak for maximal differentiation, and reconstituted as before. In both experiments nitrate reductase was produced from both fractions. The capacity of the light and the heavy proteins for reconstitution was in a ratio of between 1.2 and 1.3: 1 in spite of a twofold difference between the experimentsin the initial proportions of the light and the heavy cytochrome c reductases. The nominal yield of nitrate reductase per unit of either cytochrome c reductase was almost threefold greater for the Bio-Gel experiment. Plants were grown without added molybdenum, with or without 1pwtungsten and with either nitrate alone or nitrate and (NH&S04. Nitrate reductase activity from plants grown with NH4+ increased 3.3-fold with and 2.6-fold without tungsten after reconstitution. Without NH4+,activity was increased 4.5-fold only from plants grown with tungsten. Cytochrome c reductase activity was abundant in plants from all four 1976

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Fraction no. Fig. 2. Change in sucrose-density-centrifugation pattern of cytochrorne c reductase activity of apoprotein preparation on reconstitution of nitrate reductase in vitro Apoprotein was mixed with a pH2.5-treated nitrate reductase preparation (see the text) or with extracting buffer alone decreased to pH2.5 as a control. The mixture (0.2ml) was centrifuged through 5ml of a 5-20% sucrose gradient at 16OOOOg.,. for 16h at 2°C. Fractions (6 drops) were subdivided and assayed for cytochrome c reductase and nitrate reductase activities. Cytochrome c reductase mixed with pH2.5-treated nitrate reductase (A); in control ( 0 ) ;nitrate reductase produced by reactivation (A); in control ( 0 ) .

treatments, but reconstitution did not occur unless either NH4+, which by-passes requirements for molybdenum (Notton et al., 1974), or tungsten, which substitutes for molybdenum (Notton & Hewitt, 1971), was provided. This result is consistent with weak serological cross-reaction obtained when both were absent (Notton et al., 1974). Reconstitution occurred when plants exposed to air temperatures below 36°C but not above 42°C were used, whereas cytochrome c reductase activity persisted. Heating extracts at 35°C destroyed ability to reconstitute faster than loss of cytochrome c reductase, indicating some differential lability. Reconstitution was used to estimate the amount of apoprotein present in a molybdenum-deficient N&+-supplemented plant by mixing an extract with excess of the molybdenum component and comparing the reconstituted activity with that of the pre-acidified activity. Reconstituted activity was 79nmol of NOz-/min per g fresh weight compared with 320nmol of NOz-/min per g fresh weight originally, indicating an apoprotein content of 25 % of the normal plant enzyme. Cytochrome c reductase activity of the apoprotein extract was 36 % of that in the preacidified nitrate reductase preparation. These values compare favourably with 24 % apoprotein estimated from inhibitor studies and 36% total cross-reacting material estimated serologically (Notton et al., 1974). G. R.acknowledges receipt of an Agricultural Research Council Studentship.

VOl. 4

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BIOCHEMICAL SOCIETY TRANSACTIONS

Hewitt, E. J. (1966) Sandand Water Culture Methods Used in the Study ofplant Nutrition, 2nd edn., pp. 430-435, Commonwealth Bureau of Horticulture, Farnham Royal Hewitt, E. J. (1975) Annu. Reo. PZant Physiof. 26,73-100 Notton, B. A. & Hewitt, E. J. (1971) Biochem. Biophys. Res. Commun. 44, 702-710 Notton, B. A., Graf, L., Hewitt, E. J. & Povey, R. C. (1974) Biochim. Biophys. Actu 364,45-58

Wray, J. L. & Filner, P. (1970) Biochem. J . 119, 715-725

Folding-Pathway Selection or Cross-Linking as the Cause of Thermal Stabilization of Yeast Invertase Conformation by its Mannan JONATHAN WOODWARD and ALAN WISEMAN Department of Biochemistry, University of Surrey, Guildford, Surrey GU2 5XH,U.K.

The function of the mannan moiety of the phosphomannan protein yeast invertase (EC 3.2.1.26) remains to be elucidated. Gascbn et al. (1968) noted the higher pH stability of the cell-wall glycoenzyme compared with the mannanless invertase found only intracellularly. Purified baker’s-yeast invertase has been separated on DEAEcellulose into fractions (in the limited range of 3 9 4 5 % mannan) whose carbohydrate content and thermal inactivation rate were correlated inversely. It was concluded that the mannan moiety stabilized the invertase protein (Arnold, 1969). On the contrary the complete removal of the mannan moiety (by exo-a-mannanase treatment) from external yeast invertase affected neither the specific activity nor the thermal stability of the enzyme at 37°C (Smith & Ballou, 1974) (but was done in the presence of stabilizer, lmg of bovine serum albumin/ml). There is a continuous spectrum of different molecular forms of invertases within the yeast protoplast, and sequential addition of mannan on to internal invertase results in the formation of external invertase (Moreno et al., 1975). On the other hand, the marked difference in the amino acid composition of the internal and external invertases (Gascbn et uf.,1968) contradicts this, suggesting that internal invertase is a metabolic dead-end product (Gascbn et af., 1973). We have confirmed and extended Arnold‘s (1969) work and that of Smith & Ballou (1974). Mannan may stabilize the enzyme by cross-linking or by controlling the folding pathway or both. Chemical or enzymic removal of mannan may be misleading if removal of cross-linking is assumed. Grade VI invertase from baker’s yeast (a partially purified enzyme) was purchased from Sigma (London) Chemical Co., London S.W.6, U.K. For the purification of internal invertase a known strain of baker’s yeast (Saccharomyces cerevisiae N.C.Y.C. no. 525) was grown from loop at 30°C for 20h in shake culture in a medium containing 4% glucose, 0.2 % (NH4)2so4 and 1 % yeast extract. Internal invertase was isolated and purified (Gascbn & Lampen, 1968). Monitoring of enzyme activity was done as described previously (Wiseman & Woodward, 1975). When very low concentrations of enzyme were assayed the Perid assay of invertase was used also (Woodward et af.,1974). Thermal-stability studies were performed at 65°C (Arnold, 1969). Total protein was measured by the procedure of Lowry et af.(1 951) with bovine serum albumin as standard, and total carbohydrate was measured by the anthrone technique of Morris (1948), as modified by Chung & Nickerson (1954), with mannan as standard. The rate constants for thermal inactivation for the various enzyme preparations are compared in Table 1. Grade VI invertase was fractionated into four separate peaks of activity, by DEAE-Sephadex A-50 column chromatography, whose carbohydrate1 protein ratios differed significantly (Fig. 1). The rate of thermal inactivation obtained for each of the four ‘isoenzymes’ is correlated inversely with its carbohydrate content (in 0-75 %range obtained). Internal invertase, possessing no mannan, had lost all its activity 1976