Eur. J. Biochem. 77, 235-241 (1977)

Ca2 -Dependent Allosteric Regulation of Nicotinamide Nucleotide Transhydrogenase from Pseudomonas aeruginosa +

Bo HOJEBERG and Jan RYDSTROM Department of Biochemistry, Arrhenms Laboratory, University of Stockholm (Received November 30, 1976)

1. The addition of Ca2 to purified nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa results in a 2 - 8-fold increase in flavin fluorescence, closely resembling that induced by 2'-AMP (adenosine 2'-monophosphate). The response to Ca2+ is sigmoidal, with a Hill-coefficient of 2.6. Reduction of thionicotinamide adenine dinucleotide (oxidised form) by NADH responds in a similar fashion to Ca2+ and 2'-AMP. 2. Mg2+ inhibits the Ca2+-dependentactivation in a competitive fashion, whereas no effect of Mg2+ is observed when the enzyme is activated with 2'-AMP. 3. The pH dependencies of the activations by 2'-AMP and Ca2+ are different. The activation by Ca2+ shows little or no dependence on pH, whereas higher concentrations of 2'-AMP are required to obtain the same activation when the pH is increased. 4. The addition of both Caz+ and 2'-AMP causes a dramatic decrease in the concentration of Ca2+ required for half-maximal stimulation. 5. A negative cooperative effect of NADP+ has been demonstrated indirectly, i.e. addition of NADP' increases the concentration of Ca2 required for half-maximal stimulation. 6. These results are interpreted as indicating the existence of separate allosteric binding sites for Ca2+ and 2'-AMP. Furthermore, Ca2+ is proposed to act as a heterotropic ligand. The results are discussed in terms of current allosteric models. +

+

Nicotinamide nucleotide transhydrogenase catalyzes the reversible transfer of reducing equivalents from NADH to NADP' (cfi [l] for a review). In contrast to the mitochondria1 enzyme the transhydrogenase from Pseudomonas aeruginosa is soluble and easily purified by affinity chromatography [2,3]. The kinetic and regulatory characteristics of the purified transhydrogenase [4,5], as well as its physicochemical properties [3,6], have been extensively studied. The enzyme appears to be a flavoprotein, presumably having FAD as its prosthetic group [6]. Electron microscopy [7] and ultracentrifugation studies [6] Abbreviations and Symbols. Ado-2',S'-P2, adenosine 2 ' 3 bisphosphate; EGTA, ethyleneglycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; sNAD', thionicotinamide adenine dinucleotide (oxidized form); s O . 5 , concentration required for half-maximal stimulation derived from Hill plots; n ~ t l l Hill , coefficient. Enzymes. Alcohol dehydrogenase (EC 1.1.1.1); L-glutamate dehydrogenase (NADP') (EC 1.4.1.4); NADPH : NAD' oxidoreductase or nicotinamide nucleotide transhydrogenase (EC 1.6.1.1); glutathione reductase (NAD(P)H) (EC 1.6.4.2); pyruvate kinase (EC 2.7.1.40); fructose 1,h-bisphosphate 1-phosphohydrolase or fructose bisphosphatase (EC 3.1.3.1 1).

indicate that the isolated homogenous enzyme forms large aggregates which are filamentous in electron micrographs [7]. The physiological significance of these aggregates has, however, been questioned [2,5]. Transhydrogenase from Ps. aeruginosa has been suggested to be allosterically regulated by various 2'-nucleotides [2, 71, including NADP(H) and 2'-AMP. Since NADP+ acts as a potent inhibitor of all transhydrogenase reactions and NAD(H) is allosterically inactive [4], reduction of NADP' by NADH is very slow in the absence of an activator [4]. It seems unlikely that this is solely due to inhibition of the enzyme by the substrate NADP', since reduction of sNAD+ by NADH does not proceed in the absence of activators [2,5]. In contrast, reduction of NAD' by NADPH exhibits sigmoidal kinetics because of the allosteric activating effect of NADPH [4]. Using comparative affinity chromatography on different immobilized nucleotides, Hojeberg et al. [2] recently showed that the allosteric binding site for effector nucleotides is distinct from the active site(s) of the transhydrogenase [2], thus indicating a true

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allosteric interaction between the regulatory and active sites of the enzyme. The allosteric interactions of the purified transhydrogenase have been studied with several nonkinetic methods [7].It was demonstrated that addition of the positive effector 2'-AMP to the enzyme results in a 4 - 5-fold increase in the quantum yield of the fluorescence of the enzyme-bound FAD [7],indicating a conformational change in the protein that influences the flavin-binding region. In addition, effects on the fluorescence polarisation of the flavin were also observed [7]. It was suggested [7] that the presence of positive effectors results in the dissociation of the large transhydrogenase aggregates into smaller oligomers. In addition to being regulated by various 2'-nucleotides, the transhydrogenase from Ps.aeruginosa also is activated in a sigmoidal fashion by Ca2+,as shown by Rydstrom et al. [8]. Furthermore, it was demonstrated that Ca2 dramatically increases the affinity of this enzyme for other effectors [8], but whether or not the presence of nucleotides is essential for this activation could not be determined [8]. It seems possible to explain this activation by Ca2+ by two alternative models : firstly, Ca2 forms a complex with NADP(H) (or 2'-AMP) and the affinity of this complex for an allosteric binding site is greater than that of the free nucleotide; secondly, Ca2+ acts as a positive effector, exerting a heterotropic effect [9] when bound to a specific Ca2+-binding site on the protein. The increased affinity of the enzyme for 2'nucleotides in the presence of Ca2+ was utilized in purification of the enzyme by affinity chromatography PI. In the present study the mechanism of regulation of transhydrogenase from Ps. ueruginosa by Ca2+ and 2'-AMP in the absence of NADP(H) was investigated. The evidence presented supports the existence of separate allosteric binding sites for Ca2 and 2'-AMP. The possible physiological role of Ca2+ as an important allosteric regulator in combination with NADP(H) is discussed. A preliminary communication of the results described here has appeared [lo]. +

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and counted in a Beckman LS-100 scintillator. It was concluded that the final preparation of transhydrogenase contained less than 1 pM CaCI2 (corresponding to less than 0.1 mol of Ca2+ per mol flavin). In the activation experiments with Ca2+ this Ca2+ concentration is about three orders of magnitude lower than that required for activation and was therefore considered to be negligible. The purified enzyme was stored at 4 'C in the presence of 0.1 M 2-mercaptoethanol and used within three weeks. Chemicals

NADH, NADPH, NAD+, NADP', 2'-AMP and EGTA were purchased from Sigma Chemicals (St Louis, Mo., U.S.A.), alcohol dehydrogenase, sNAD +,oxidized glutathione and glutathione reductase from Boehringer (Mannheim, Germany) and 45CaC12 was obtained from Radiochemical Centre (Amersham, England). Other chemicals were of reagent grade. Enzyme Assays

Reduction of sNAD' by NADH was followed in an Aminco-Chance DW spectrophotometer at 400 nm, assuming an absorption coefficient for sNADH of 11.3 x lo3 M-' cm-' [ll]. The assay medium contained 0.5 mM NADH and 0.5 mM sNAD+ in 0.1 M Tris-C1 buffer (pH 7.0); added effectors were present at concentrations indicated in the legends. Reduction of NADP' by NADH was followed as described earlier [ 121, using glutathione reductase and oxidized glutathione as a regenerating system for NADP'. All enzyme activities were measured at 30 "C. Flavin Fluorescence

Flavin fluorescence was monitored in an AmincoBowman spectrofluorometer in the ratio mode, at room temperature. The excitation wavelength was 365 nm and the emission wavelength was 510 nm; 1-mm slits were used. RESULTS

MATERIALS AND METHODS Transhydrogenuse

Transhydrogenase from Ps.aeruginosa was purified as described earlier [2]. The purification procedure requires the presence of CaC12 in the affinity chromatography step, and a pulse of 45CaC12was included in the buffer prior to elution from the affinity column with EDTA and higher pH. After a subsequent gel filtration step on a Sephadex G-200 column, samples of the purified enzyme were added to Brays' solution

Effect of Ca2+on Transhydrogenase Fluorescence and Activity

Louie et al. [7] demonstrated that a conformational change in the transhydrogenase from Ps.aeruginosa induced by addition of the positive effector 2'-AMP may be monitored by the concomitant 4- 5-fold increase in the fluorescence of the enzyme-bound flavin. The possibility that Ca2 could replace 2 '-AMP in inducing this fluorescence change in the purified transhydrogenase was investigated. As demonstrated in Fig. 1, Ca2+ does indeed cause a 2-8-fold increase +

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in the quantum yield of the fluorescence, both for emission (Fig. 1A) as well as for excitation (Fig. 1B). This effect, analogous to that of 2'-AMP [7], indicates a Ca2 -dependent allosteric conformational change. No effect of Ca2+ on the visible absorption spectrum of the purified transhydrogenase could be seen (not shown). The fluorescence enhancement varied between different preparations. Moreover, it decreased considerably as the enzyme preparations aged. This effect may be attributed primarily to an increase in the basal fluorescence, rather than to a decrease in the fluorescence in the presence of Ca2+. The decrease in fluorescence enhancement was accompanied by a loss of the absolute requirement for an activator in the reduction of sNAD+ by NADH by the transhydrogenase. Consequently, only preparations showing at least a 4-fold increase in fluorescence upon addition of a positive effector were used in this study. The response of the flavin fluorescence of the transhydrogenase upon addition of increasing amounts of either Ca2+ or 2'-AMP is sigmoidal, giving Hill plots with slopes exceeding the value of 2 (Fig. 2A and B). This finding indicates the existence of at least two separate but interacting binding sites for each effector. In addition, Fig. 2 shows the allosteric effects of 2'AMP and Ca2+ on the reduction of sNAD+ by NADH. This reaction is suitable for selective studies of the influence of Ca2+ on the transhydrogenase, since 2'-nucleotides are not required as substrates or effectors. By comparing the results obtained by fluorometric and kinetic titrations, it is seen (Fig. 2) that the S5.5 as well as the nHil1 for the two effectors are similar, although a slight discrepancy between the So.5 obtained by the different methods is seen. These results indicate that the sigmoidal changes in fluorescence of the enzyme-bound flavin induced by effectors is related to the allosteric conformational changes of the protein. The differences in So.5 values obtained by different techniques could be the result of difficulties in obtaining a true value for V. Alternatively, a small non-specific activation may be caused by one or both of the substrate nucleotides used, since this phenomenon was noticed both with Ca2+ and 2'AMP.

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Fig. 1. Eflect ofCa2+ on the flavin fluorescence spectrum ojpurified transhydrogenase. ( A )Emission spectrum, A,, = 365 nm. (B) Excitation spectrum, A,, = 510 nm. The concentration of the enzymebound flavin was 50 nM, with a specific activity of 0.8 pcat/mg protein in 0.1 M Tris-C1 (pH 7.0) and 0.1 M 2-mercaptoethanol

In a previous paper [2] the dramatic effect of pH on both the So.5for free 2'-nucleotides, as well as on the binding capacity of transhydrogenase to the immobilized effector Ado-2',5'-P2, was demonstrated. An increase in pH results in both a large increase in the So.5 for the free effector and a decreased affinity for the immobilized nucleotide. In order to study further the detailed mechanism of regulation by Ca2+ and 2'-AMP, the activation of sNAD+ reduction by

238

Allosteric Regulation of Pseudoomonus Transhydrogenase

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[ 2’- AMP] ( KM) Fig. 2. Hill plot of the allosteric uctivution qf tmiishyM’rogc.viase hy Cu2- [ A ) ur 2‘-AMP ( E ) . (0-0) Increased emission fuorescence at 510 nm, I,, = 365 nm. (0--0) Activation of reduction of s N A D + by NADH (cf: Methods). All experiments in (B) were performed in the presence of 1 mM EGTA

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NADH by these effectors was studied at different pH values. As indicated in Fig. 3A, the effects of these substances are distinguishable in that the So.5 for Ca2+ is essentially unaffected by pH, whereas the S0.5 for 2‘-AMP increases with pH. This is in agreement with earlier results obtained with Ado-2’,5’-P2 [2]. These results were extended by performing the corresponding titrations of the flavin fluorescence (Fig. 3B). Effect of M2’

on the Activation by Ca2’

It has been reported earlier [8] that Mg2+ inhibits Ca2+-dependentactivation of the transhydrogenase. In order to further elucidate the differences between the mechanism of regulation by Ca2+ and 2‘-AMP, the effect of Mg2+ on the activation by these effectors was investigated. As demonstrated in Fig. 4, Mg2+

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Fig. 3. CJJrct o f p H on thr a l h t e r i c activation of rransh,vdru~enuse h,v Cuz and ? ‘ - A M P . In (A) the transhydrogenase-catalyzed reduction of s N A D + by N A D H was followed as described in Materials and Methods. In (B) the enhanced flavin fluorescence was monitored; except for pH experimental conditions were as in Fig. 1 A. In the case of 2’-AMP activation, 1 mM EGTA was present. ( 0 - 0 ) Ca2 ‘ -dependent activation; (0---o)2’-AMP-dependent activation +

239

B. Hojeberg and J. Rydstrom 10 000

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Fig. 5. Comhined ejji>cts qf Ca2 and 2'-AMP on the enhancement in the flavin fluorescence of the transhydrogenase. Experimental conditions as in Fig. 1A

Negative Coopevativity Induced by NADP'

Fig. 4. Effect of M g 2 + on the enhancement in the fluvin fluorescence of the transhydrogenuse. (.--a) Caz+-dependent increase in fluorescence. (0--0)2'-AMP-dependent increase in fluorescence. Experimental conditions as in Fig. 1A

inhibits Ca2+-activation in a competitive manner when enhanced flavin fluorescence is measured. In contrast, the So.5 for 2'-AMP seems to be essentially unaffected by the concentrations of Mg2'used (Fig. 4). It should be noticed that in the absence of Ca2+ no effect of Mg2+ on the flavin fluorescence spectrum was observed, in agreement with the conclusion that Ca2+ rather than Mg2 is the allosterically active metal ion [8]. +

Ejjhct of 2'-AMP on the Activation by Ca2i 2'-AMP has been the most frequently studied effector of the various transhydrogenase reactions. The activating effect of this nucleotide is probably due to its structural resemblance to NADP(H). It was reported earlier [8] that Ca2+ strongly influences the So.5 for both 2'-AMP and NADPH, but the nature of this effect was not fully explored. In Fig. 5 it is demonstrated that the addition of 2'-AMP dramatically affects the for Ca". This type of dependency appears to be incompatible with the possibility that a complex of Ca2+ and 2'-AMP is the allosterically active effector of the transhydrogenase. The marked effect of 2'-AMP on the Ca2+-dependent activation illustrates the potency with which the enzyme may be regulated by various 2'-nucleotides at suboptimal levels of CaZ . +

NADP' is known to inhibit the transhydrogenase activity [5], and it was suggested [4] that this inhibitory effect could be due to either the formation of a deadend complex with the enzyme or an allosteric inhibition. In order to clarify the mechanism of this inhibition of transhydrogenase by NADP' , the concentration of Ca2+ was varied in the presence of varying concentrations of NADP' . The negative cooperative effect of NADP' is demonstrated indirectly in Fig. 6 for both the enhancement in flavin fluorescence and the reduction of NADP' by NADH. Fig. 6 A shows the effect of increasing amounts of NADP' on the So.5 for Ca2+ in the enhancement of flavin fluorescence. Similar results were obtained for reduction of NADP' by NADH at various concentrations of NADP' using glutathione reductase as the regenerating system (Fig. 6B). This negative cooperative effect of NADP' is mediated by the regulatory site specific for 2'-nucleotides, appears to be homotropic, and satisfactorily explains why NADP' acts as a potent inhibitor of the enzyme [4]. These findings are not inconsistent with the postulate [2,5] that NADPH (and 2'-AMP), which shows a positive cooperative effect [4], also binds to the regulatory site specific for 2'-nucleotides.

DISCUSSION The present data strongly support previous proposals [2,8] that Ca2+ is a potent positive effector of nicotinamide nucleotide transhydrogenase from Pseudomonas aevuginosa. Several lines of evidence presented in this paper, obtained mainly from nonkinetic studies of the enzyme-bound flavin as well as from direct kinetic analysis of reduction of sNAD+

Allosteric Regulation of Pseudomonas Transhydrogenase

240

Active site

Allosteric sites

NAD(P)' NAD(P)H

Fig. 7. Schematic representation of different binding sites of transhydrogenase

in combination with Ca2+ are responsible for the allosteric regulation seen with Ca2+.The results thus strongly support the hypothesis [S] that free Ca2+, rather than a hypothetical Ca2 - 2'-nucleotide complex, is the true allosteric effector of transhydrogenase, since the complex would be expected to exhibit a pH dependency similar to that obtained with 2'-AMP only. The discrepancy observed in the pH dependency of SO5 for Ca2+ and 2'-AMP could be an indication that the protonated species of the 2'-phosphate are the allosterically active forms of the nucleotide. Alternatively, this observation may also be the results of differences in the pK, values of the amino acid residues involved in the binding of the two types of effectors. A schematic representation of the different binding sites of transhydrogenase is shown in Fig. 7. Generally, enzyme mechanisms showing negative homotropic cooperativity of ligand binding in nonkinetic systems are incompatible 1141 with the symmetry model of Monod et al. [9] for allosteric transitions, but are compatible with the sequential model of Koshland and coworkers [15,16]. A finding pertinent to this point is that NADP' influences the Ca2+enhanced flavin fluorescence of the transhydrogenase in a negative cooperative manner. These results, which are confirmed by the kinetic data, seem therefore to rule out the model of Monod et al. [9] in the case of the allosteric transitions of the transhydrogenase. Louie et al. [7] have demonstrated that the Pseudomonas transhydrogenase undergoes dissociation upon addition of 2'-AMP. It was proposed that this phenomenon reflects the allosteric change in the enzyme conformation, although the transition was an all-or-none rather than an equilibrium process [7]. Recently, Wermuth and Kaplan [3] argued against this hypothesis, and demonstrated that the two forms of the enzyme both require 2'-AMP for full activity. These results are in agreement with our findings that neither 2'-AMP nor Ca2+ are capable of dissociating the enzyme into smaller subunits, although the enzyme was shown to be allosterically activated by the fluores+

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Fig. 6 . Effect of NADP' on enzymatic activity ( A ) andenhancement in flavin fluorescence ( B ) of the Ca2+-activated transhydrogenase. In (A) the fluorescence increase was followed as described in Fig. 1 A. In (B) reduction of NADP' by NADH was followed at various [NADP'], using glutathione reductase as the regenerating system, as described in Materials and Methods. [NADH] was 200 pM in 0.1 M Tris-Cl (pH 8.0)

by NADH in the absence of 2'-phosphonicotinamide nucleotides, favour the existence of a Ca2+-specific allosteric binding site in the enzyme. The data suggest, moreover, that Ca2+ acts as a heterotropic ligand. However, all reactions catalyzed by the transhydrogenase take place readily in the presence of saturating concentrations of 2'-AMP and high concentrations of EGTA, implying that Ca2+ is not an essential cofactor of the enzyme. The transhydrogenase from Ps. aeruginosa thus appears to be different from many other enzymes that are allosterically activated by divalent metal ions [13]. Additional support for a Ca2+-specific binding site emerges from the data obtained with binding of Mg2+ to the enzyme. This binding leads to a competitive exclusion of Ca2+ from the metal-ion-binding site without affecting the 2'-AMP-dependent activation. The difference in the pH dependencies of the for Ca2+ and 2'-AMP appears to eliminate the possibility that contaminating amounts of 2'-nucleotides

241

B. Hojeberg and J. Rydstrom

cence technique (B. Stensland, R. Norrestam and B. Hojeberg, unpublished observations). Thus, at present it is difficult to draw any conclusion regarding a correlation between dissociation and allosteric conformational changes induced by effectors. Ca2+ is known to be an allosteric activator of other enzyme systems [17,18]. In the case of pyruvate kinase from human erythrocytes Ca2+ was suggested to bind to different forms of the enzyme, thereby effecting the mechanism of catalysis [17]. Furthermore, glutamate dehydrogenase from Lemna minor responds in a sigmoidal fashion to Ca2+ in the NAD(H)dependent reaction, but not in the NADP(H)-dependent reaction [18]. It has been suggested that the native form of fructose-l,6-biphosphatasefrom beef liver is allosterically regulated not only by AMP, but also by Mn2+ and Mgz+.However, the enzyme has a dual requirement for divalent metal ions, which are involved in binding to both effector and catalytic sites ~31. Kaplan and coworkers recognized early the apparent lack of reversibility of reduction of NAD' by NADPH catalyzed by the Pseudomonas transhydrogenase. On the basis of this finding it was postulated [19] that the biological role of the transhydrogenase is to transfer reducing equivalents from NADPH (generated, for instance, by the NADP-dependent isocitrate dehydrogenase reaction) to NADH and subsequently into the respiratory chain [19]. This would be opposite to the mitochondria1 enzyme, where respiratory energy or ATP is utilized to drive the reaction towards NADPH production [20]. In view of the findings reported here it appears likely that under biological conditions Pseudomonas transhydrogenase may operate as a truly reversible transhydrogenase, especially at pH values below 7, where the inhibitory effect of NADP' is decreased (B. Hojeberg and J. Rydstrom, unpublished results). Although the physiological concentration of free Ca2+is usually of the order of pM [21], it seems likely that the transhydrogenase is regulated by the prevailing redox level of NADP in combination with small changes in the intracellular concentrations of nonchelated Ca2+,as well as by changes in pH. The concentration of free Ca2' in turn is regulated by several factors, including indirectly the energy state of the cell [21]. Since ATP chelates Ca" with a 30-fold higher

affinity than ADP [22], these factors include the ATP/ADP ratio and the steady-state concentrations of intermediate tricarboxylic acids. In addition, the bacterium may regulate transhydrogenase activity by accumulating Ca2 from the surrounding medium. +

This work was supported by the Swedish Cancer Society (102-137-10XA).

REFERENCES 1. Rydstrom, J., Hoek, J. & Ernster, L. (1976) in The Enzymes (Boyer, P. D., ed.) 3rd edn, vol. 13, pp. 51-88, Academic Press, New York. 2. Hojeberg, B., Brodelius, P., Rydstrom, J. & Mosbach, K. (1976) Eur. J . Biochem. 66,467-475. 3. Wermuth, B. & Kaplan, N. 0. (1976) Arch. Biochem. Biophys. 76, 136-143. 4. Cohen, P. T. & Kaplan, N. 0. (1970) J . Biol. Chem. 245, 4666 - 4672. 5. Widmer, F. & Kapkdn, N. 0. (1976) Biochemistry, 15, 46934698. 6. Cohen, P. T. & Kaplan, N. 0. (1970) J . Biol. Chem. 245, 2825-2836. 7. Louie, D. D., Kaplan, N. 0. & McLean, J. D. (1972) J . Mol. Biol. 70, 651 - 664. 8. Rydstrom, J., Hoek, J. & Hojeberg, B. (1973) Biochem. Biophys. Res. Commun. 52, 421 429. 9. Monod, J., Wyman, J. & Changeux, J. P. (1965) J . Mol. Bid. 12, 88-118. 10. Hojeberg, B. & Rydstrom, J. (1976) Abstr. Commun. IUth. Meet. I n / . Union Biochem. no. 07-4-117. 11. Pabst Laboratories (1961) Ultraviolet Absorption Spectra of Pyridine Nucleotide Coenz.ymes and Coenzyme Analogues, Pabst Laboratories circular OR-18. 12. Rydstrom, J. (1977) Mefhods Enzymol. in the press. 13. Nimmo, H. G. & Tipton, K. F. (1975) Eur. J . Biochem. 58, 575-585. 14. Goldbetter, A. (1974) J . M o l . Biol. 90, 185-190. 15. Koshland, D. E., Nemethy, G . & Filmer, D. (1966) Biochemistry, 5, 365- 385. 16. Koshland, D. E. (1970) in The Enzymes (Boyer, P. D., ed.) vol. 1, pp. 341 -396, Academic Press, New York. 17. Flikweert, J. P., Hoorn, R. K. J. & Staal, G. E. J. (1975) Biochemie, 57,677-681. 18. Hartmann, T. & Ehmke, A. (1976) Ahstr. Commun. 10th Meet. Int. Union Biochem. no. 07-5-109. 19. Widmer, F. & Kaplan, N. 0. (1976) Riochcmi.sfry, 15; 46994702. 20. Rydstrom, J. (1977) Biochim. Biophys. Arta, in the press. 21. Kretsinger, R. H. (1976) Annu. Rev. Biochem. 45, 239-266. 22. Data .for Biochemical Research (1972) (Dawson, R. M. C., Elliot, D. C., Elliot, W. H. & Jones, K. M., eds) 2nd edn, pp. 433-434, Oxford University Press, London.

B. Hojeberg and J. Rydstrom, Avdelningen for Biokemi, Arrhenius La bordtoriet, Stockholms Universitet, Fack, S-106 91 Stockholm, Sweden

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