Photosynthesis Research 26: 161-170, 1990. © 1990 KluwerAcademic Publishers. Printedin the Netherlands. Regular paper

Oligomeric enzymes in the

C4

pathway of photosynthesis

Florencio E. Podestfi 1, Alberto A. Iglesias 2 & Carlos S. Andreo 3 Centro de Estudios Fotosintdticos y Bioquimicos. Suipacha 531. 2000 Rosario; 1Present address: Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6; 2 Present address: Department of Biochemistry, 201 Biochemistry Building, Michigan State University, East Lansing, Michigan 48824-1319, USA; 3 To whom all correspondence should be addressed Received 2 March 1990; accepted 2 August 1990

Key words: NAD-malic enzyme, NADP-malic enzyme, NADP-malic dehydrogenase, pyruvate, phosphate dikinase, regulatory protein, phosphoenolpyruvate carboxylase, protein structure, enzyme regulation, C 4 metabolism Abstract

This review deals with the factors controlling the aggregation-state of several enzymes involved in C 4 photosynthesis, namely phosphoenolpyruvate carboxylase, NAD- and NADP-malic enzyme, NADPmalic dehydrogenase and pyruvate, phosphate dikinase and its regulatory protein. All of these enzymes are oligomeric and have been shown to undergo changes in their quaternary structure in vitro under different conditions. The activity changes linked to variations in aggregation-state are discussed in terms of their putative physiological role in the regulation of C 4 metabolism.

Abbreviations: P-enolpyruvate-phosphoenolpyruvate; N A D - M E - N A D - d e p e n d e n t malic enzyme; N A D P - M E - N A D P - d e p e n d e n t malic enzyme; N A D P - M D H - N A D P - d e p e n d e n t malic dehydrogenase; P P D K - p y r u v a t e , phosphate dikinase; P P D K - R P - pyruvate, phosphate dikinase regulatory protein; Vmax - m a x i m a l velocity; Kin,- Michaelis constant; C A M - Crassulacean acid metabolism

Introduction

The regulation of carbon flux in higher plants is a process requiring the concerted modulation of a large number of enzymes through a wide variety of mechanisms such as reversible protein phosphorylation (Budde and Chollet 1988, GuidiciOrticoni et al. 1988), changes in the redox state of certain -SH residues (Buchanan 1980, Anderson 1979, Edwards and Walker 1983) and control of activity through metabolite levels (Edwards and Walker 1983, Cs6ke et al. 1984, Woodrow and Berry 1988). Many of these changes are elicited by light-dark transitions, and thus make possible the movement of carbon skeletons in

the 'right' direction according to the plant's requirements. One of the factors that can dramatically change the kinetic properties of enzymes is the degree of polymerization. Many proteins participating in carbon assimilation pathways have been shown to undergo association-dissociation in vitro. These include NAD-malic enzyme from Solanum tuberosum (Grover and Wedding 1984) and Crassula (Willeford and Wedding 1986); maize leaf pyruvate, phosphate dikinase (Edwards et al. 1985) and its regulatory protein (Edwards et al. 1985); P-enolpyruvate carboxylase from CAM (Jones et al. 1978, Wu and Wedding 1985, 1987) and C 4 (Walker et al.

162

i

_~

2

Chloroplast

C(

L

]

dr,onl

sp

t

PEP

~

~~RP~/ppoK, ~,, ,~.Pyr

----..-

. ~ -

_ . _ _ l

_

Bundle sheath cells

~

Chloroplast

~. _ _~~c3Chloroplast

Mesophyllcell

__~~c

co~

Fig. 1. Schematic representation of C 4 metabolism showing the enzymes that could be subject to regulation through changes in oligomeric state. PPRP: Pentose phosphate reductive pathway.

Table 1. Summary of the observed molecular weights and location of oligomeric enzymes of C 4 metabolism Enzyme

Location ~

Subunit mol. wt (kDa)

PEPC

M Cyt

I00

Observed aggregation state

Reference(s)

O'Leary 1982, Andreo et al. 1987, Stiborov~ 1988

M

(active) (partially active) (inactive)

T D

PPDK

M Chl

96

T D

(active) (inactive)

Edwards et al. 1985

PPDK-RP

M Chl

45

T D

(active) (active)

Edwards et al. 1985; Burnell and Hatch 1983, 1985

NADP-MDH

M Chl

37-43

T D M

(active) (active) (inactive)

Edwards et al 1985

NADP-ME

BS Chl

62.5

T D M

(active) (active) (active)

Thorniley and Dalziel 1988, Iglesias and Andreo 1990a,b

NAD-ME*

BS Mit

49-67

0 T D M

(active?) (active) (inactive) (inactive)

Artus and Edwards 1985 Wedding 1989

For reference on location see Edwards and Walker 1983. M Cyt: mesophyll cytoplasm; M Chh mesophyll chloroplast; BS Chh bundle sheath chloroplasts: BS Mit: Bundle sheath mitochondria; O: octamer; T: tetramer; D: dimer; M: monomer. * Data corresponding to the C 3 enzyme.

163 1986a,b, Wagner et al. 1986, 1987, 1988) plants; NADP-dependent malic dehydrogenase from pea (Fickenscher and Scheibe 1983, Edwards et al. 1985) and maize (Edwards et al. 1985); and the NADP-dependent malic enzyme from sugarcane (Iglesias and Andreo 1990). In most cases, the oligomeric state of these enzymes depends on ionic strength, pH, the redox state, temperature, protein concentration and the presence of cosolutes and metabolites (substrates or allosteric effectors). Although many of the characteristics of these polymerization reactions have been described in recent years, the actual physiological role of polymerization still remains an open question. In fact, the experimental conditions generally used are very different from those existing within the plant cell, particularly with respect to the in vivo protein concentration (Fulton 1982). However uncertain, the assignment of a physiological role for changes in quaternary structure seems very attractive in view of its great impact on enzyme activity, especially when it is taken into account that many of the control mechanisms cited above are also important for the maintenance of aggregation state (Frieden 1989). Since most of the enzymes mentioned above are involved in the C 4 pathway of CO2 fixation (Fig. 1 and Table 1), we thought it was of interest to present a short review dealing with the recent advances in the study of polymerization of various C 4 enzymes and the possible relation with the regulation of carbon metabolism in this type of plant.

Phosphoenolpyruvate carboxylase P-enolpyruvate carboxylase (E.C. 4.1.1.31) is the protein responsible for the initial carboxylation step in C 4 plants (Uedan and Sugiyama 1976) (Fig. 1). The reaction, which proceeds via a two-step mechanism (Gonzfilez and Andreo 1989), involves the carboxylation of the threecarbon substrate P-enolpyruvate with HCO3 to yield oxaloacetate and orthophosphate as products. The protein has often been described as a homotetramer of 400kDa (O'Leary 1982, Andreo et al. 1987). Most studies on changes in the

quaternary structure of this carboxylase have been conducted using the CAM enzyme (Wu and Wedding 1987, Krfiger and Klfige 1987). However, the possible significance of the oligomeric state of P-enolpyruvate carboxylase in the physiological regulation of CAM metabolism is still a matter of debate (Kr/iger and Klfige 1987). The C 4 leaf enzyme has been shown to undergo dissociation into largely inactive dimers and monomers upon chemical modification of histidyl and sulfhydryl groups (Walker et al. 1986b), a process prevented by the presence of one of the substrates, P-enolpyruvate, and Mg 2+. The carboxylase is also susceptible to increasing ionic strength (Manetas et al. 1986, Stiborovfi and Leblovfi 1987, Wagner et al. 1986, 1987, 1988) and in the presence of NaC1 it dissociates into dimers at pH7.0 or dimers and monomers at pH 8.0 (Stiborovfi and Leblovfi 1987, Wagner et al. 1986, 1987, 1988). The salt-induced dissociation is also prevented by the presence of Penolpyruvate and Mg 2+ or glucose-6-phosphate (an allosteric activator) (Wagner et al. 1986, 1987). High protein concentrations (Wagner et al 1987, Selinioti et al. 1987, McNaughton et al. 1989), cosolutes (Selinioti et al. 1987) and glycerol (Podestfi and Andreo 1989) also shift the equilibrium to the larger tetrameric form. However, glycerol does not protect P-enolpyruvate carboxylase against NaCl-induced dimerization (Podestfi and Andreo 1989). The dominant effect of dissociation is an increase in the K m for P-enolpyruvate (Wagner et al. 1986, 1987, Podestfi and Andreo 1989). The 200-kDa dimeric form obtained upon saltinduced dissociation has been reported to be either partially active (Wagner et al. 1986, 1987), although to a much lesser extent than the tetramet (about 20%), or inactive (Chou et al. 1986, Walker et al. 1986b, Stiborovfi and Leblovfi 1987). The monomer appears to be totally inactive (Wagner et al. 1986, 1987, Walker et al. 1986b). It is worth noting that chemical modification studies with fluorescent probes followed by fluorescence energy transfer measurements suggest that each subunit contains at least one substrate binding site (Wagner et al. 1988). The inhibitor L-malate has been shown to produce partial enzyme dissociation into dimers (McNaughton et al. 1989; Podestfi and Andreo,

164 unpublished results). Huber et al. (1986), however, found that L-malate acted to preserve the different characteristics of the day and night forms of P-enolpyruvate carboxylase, while producing only subtle changes in its native molecular weight. Presumably this occurs by distorting the enzyme's tertiary rather than the quarternary structure. Conditions which promote enzyme dissociation (i.e., pH near 7.0, NaCI, absence of dithiol reductants) induce sigmoidal kinetics for P-enolpyruvate binding (Uedan and Sugiyama 1976, Karabourniotis et al. 1983, Stamatakis et al. 1988, Stiborovfi 1988, Podestfi and Andreo 1989). Interestingly, under conditions where most of P-enolpyruvate carboxylase protein should be in its tetrameric form (i.e., pH8.0, high P-enolpyruvate-Mg 2+ levels, plus glycerol, plus proline or betaine) the response of activity to increasing P-enolpyruvate concentrations is hyperbolic (Uedan and Sugiyama 1976, Manetas et al. 1986, Stamatakis et al. 1988, Stiborovfi 1988, Podest~i and Andreo 1989). It is, therefore, tempting to speculate that changes in P-enolpyruvate carboxylase quarternary structure could be at least partially involved in the control of activity during light-dark transitions. However, at present there is no experimental evidence in support of this speculation (Budde and Chollet 1986, Nimmo et al. 1986 McNaughton et al. 1989, Huber et al. 1986) Much work should be done to test this hypothesis, and it will be a difficult task to unequivocally prove it. First, the roles of glucose-6-phosphate and malate on the polymerization process must be thoroughly studied. Second, precise measurements of the metabolite levels and pH in the mesophyll cytoplasm should be made available. Finally, molecular weight and activityresponse measurements should be performed under conditions mimicking more accurately the in vivo conditions (i.e., high protein concentration in the presence of a mixture of the various P-enolpyruvate carboxylase effectors at their physiological concentrations and pH). However, the protein concentration that can be used in this kind of experiment in thought to be very different from that actually existing in the plant cell (Fulton 1982). Protein phosphorylation could also be closely related with the oligomeric inter-

conversion (Frieden 1989), since it sharply changes the enzyme's response to its metabolic effectors (Nimmo et al. 1987, Jiao and Chollet 1988). Although presenting a somewhat different pattern of regulation, the structural studies on the CAM enzyme may provide some clues for the understanding of the oligomeric interconversion of C a P-enolpyruvate carboxylase. The Crassula enzyme has been postulated to exist in a dimeric/ malate-sensitive form during the day and as a tetrameric/malate-insensitive form during the night (Wu and Wedding 1985, 1987). The latter is considered as functional during nocturnal CO 2 assimilation (Wu and Wedding 1987). Malate shifts the equilibrium towards the dimer, while preincubation with P-enolpyruvate favors the tetramer of P-enolpyruvate carboxylase (Wu and Wedding 1987). A strict correspondence of the phosphorylation state of the CAM enzyme with its aggregation state is not yet evident. Contrasting with the above reports, the day and night forms of the enzymes from Bryophyllum fedtschenkoi (Nimmo et al. 1986) and Kalanch6e daigremontiana (Kriiger and Klfige 1987) were found to be in a single aggregation state (tetramer for the first species and dimer for the second).

NAD-malic enzyme

NAD-malic enzyme (NAD-ME, E.C. 1.1.1.39) oxidatively decarboxylates malate in the mitochondrion, yielding NADH, CO 2 and pyruvate as products. It is responsible for the decarboxylation step in the NAD-ME subgroup of C 4 plants (Edwards and Walker, 1983) (Fig. 1), but it also plays an important role in the P-enolpyruvate carboxykinase subgroup, providing much of the ATP necessary for the carboxykinase-catalyzed decarboxylation of oxaloacetate and also part of the CO 2 to be fixed through the C 3 photosynthetic carbon reduction cycle (Burnell and Hatch 1988). Despite its importance in C 4 plants, little effort has been dedicated to studying the enzyme from this source (Artus and Edwards 1985). For this reason, most of the studies cited here have been carried out with the enzyme from C 3 or CAM plants. However, the marked simi-

165 larity between NAD-MEs suggests that it is reasonable to extend these findings to the C 4 enzyme (Hatch et al. 1974, Wedding 1989). NAD-ME from various sources has been found as a dimer, tetramer and octamer (Artus and Edwards 1985), with a subunit mass ranging from 49 to 67kDa (Willeford and Wedding 1987a). A heterogeneous composition of the oligomers of plant NAD-ME could be the result of proteolysis of the original subunit or may indicate the presence of isoenzymic polypeptides. Willeford and Wedding (1987a) addressed this question and postulated that plant NAD-ME is composed of two different subunits, each necessary for enzymic activity and being of a different nature (i.e., one is not derived from the other as expected from a proteolytic transformation). This seems to be a characteristic of only plant NAD-MEs since the microbial or animal enzymes are aggregates of a single polypeptide (Wedding 1989). The catalytic activity of plant NAD-ME is dependent on its quaternary structure (Grover and Wedding 1984, Artus and Edwards 1985, Willeford and Wedding 1986, Wedding 1989). It has been suggested that the octamer-tetramer, rather than the tetramer-dimer equilibrium is the one associated with the control of activity (Artus and Edwards 1985). The enzymatic forms with the larger molecular weight (octamer and/ or tetramer) are perhaps the most active. The equilibration between oligomeric forms is rapid (Willeford and Wedding 1986). The stability of the enzyme depends on protein concentration (Grover and Wedding 1982), ionic strength (Grover and Wedding 1982) and the presence of reducing agents (Macrae 1971, Dittrich 1976) or the activator Coenzyme A (CoA) and/or malate (Willeford and Wedding 1987b). A change in the oligomeric state alters the K m (malate) and the degree of cooperativity for substrate binding (Artus and Edwards 1985, Wedding 1989). The in vivo regulation could be accomplished by malate-mediated changes in the aggregationstate (Wedding 1972), CoA, fumarate, the Mn2+/Mg 2+ ratio (Grover et al. 1981, Willeford and Wedding 1986), and/or pH (Willeford and Wedding 1987b). As Wedding (1989) notes in a recent review, the variations in kinetic properties induced by these effectors, as well as the lag of

activity, may reflect quarternary structure changes occurring during assay. Although conclusive evidence may arise in the future, the work performed at present indicates that NADME is certainly one of the most promissory for performing structure-based regulation.

NADP-dependent malic enzyme The NADP-dependent malic enzyme (NADPME, E.C. 1.1.1.40) catalyzes the oxidative decarboxylation step of C 4 metabolism in the bundle sheath chloroplasts in maize, sugarcane and other C 4 plants (Edwards and Walker 1983) (Fig. 1). Previous work indicates that the enzyme exists mainly as a homotetramer of 220-260 kDa (Asami et al. 1979, Hausler et al. 1987, Iglesias and Andreo 1989). The aggregation state of maize NADP-ME is dependent on the presence of a dithiol as well as the buffer used (Thorniley and Dalziel 1988). The sugarcane enzyme may exist in both a tetrameric and dimeric form in solution, depending on pH (Iglesias and Andreo 1990a). At pH8.0, where NADP-ME displays greater activity (higher V~ax, lower K m for Lmalate and NADP + and a significantly higher affinity for Mg 2+) it is found mostly as a tetramer of 250 kDa, whereas at pH 7.0 (with lower Vmax, higher K m for L-malate and NADP + and inhibition by high L-malate) the dimeric form is predominant (Iglesias and Andreo 1990a). Both oligomers are readily interconvertible (Iglesias and Andreo 1990a) and they are active, although with different catalytic efficiency (Iglesias and Andreo 1990b). High ionic strength dissociates the enzyme to active dimers and monomers (Iglesias and Andreo 1990b). Mg 2+ also affects the dimer-tetramer equilibrium, inducing the aggregation of the protein (Iglesias and Andreo 1990b). Interestingly, pH and Mg 2+ levels, which mainly affect oligomerization of NADP-ME, change in the chloroplast stroma (the site where the enzyme is located, Edwards and Walker 1983) in light or dark periods (Heldt 1979). Although, as in the case of P-enolpyruvate carboxylase, further studies could provide the evidence necessary to confirm whether these structural changes are linked or not with the in vivo control of NADP-ME activity, a model for

166 the light-dark regulation of this chloroplastic decarboxylation reaction based on the available information would be as follows: i) dark stromal conditions (pH7.0, low Mg 2+) would cause dissociation, rendering an enzyme form with low affinity for NADP + and Mg 2+ and sensitive to inhibition by excess L-malate; ii) light conditions in the stroma (pH 8.0, high Mg 2+) would favor association, which in turn renders an enzyme form with higher Vmax and affinity for NADP + and Mg 2+

NADP-malate dehydrogenase In the NADP-ME subgroup of C 4 plants the mesophyll chloroplast reduction of the oxaloacetate formed by the initial carboxylation reaction in the cytoplasm is catalyzed by an NADPdependent dehydrogenase (NADP-MDH, E.C. 1.1.1.82) (Fig. 1), which uses the N A D P H produced by photosynthetic electron transport (Edwards and Walker 1983). Although the maize enzyme has been extensively studied, persistent differences have remained regarding its molecular weight. Johnson and Hatch (1970) found three peaks of activity after size-exclusion chromatography, with most of the activity located at a 150-kDa peak. Kagawa and Hatch (1977) found that NADP-MDH activation through reversible dithiol reduction is followed by dimerization of the inactive monomer. Ashton and Hatch (1983) and Kagawa and Bruno (1988) reported the redox-activated and inactivated forms of the enzyme as having identical molecular weights, but while the first authors found only a tetrameric, 150-kDa form, the latter observed a dimeric one of around 90 kDa. Limited proteolysis of the inactive (i.e., oxidized) tetrameric C 3 enzyme from pea chloroplasts (Fickenscher and Scheibe 1988) results in the formation of active dimers less prone to reduction by thioredoxin. Whether the same is true for the maize dehydrogenase is unclear, especially in light of the report by Kagawa and Bruno (1988) that endogenous proteases reduce the monomeric molecular weight from 43 to 37 kDa and partially inactivate NADP-MDH. In

fact, proteolysis could be one of the reasons for the many discrepancies regarding subunit and oligomer molecular weights of the C 4 dehydrogenase. A discussion on the possible involvement of polymerization reactions in the regulation of NADP-MDH at this time is not worthwhile because of the many contradictory reports.

Pyruvate, phosphate dikinase Pyruvate, phosphate dikinase (PPDK, E.C. 2.7.9.1) is an oligomeric enzyme, composed of four identical subunits, responsible for the regeneration of the primary substrate of C 4 carboxylation, phosphoenolpyruvate, in the C4mesophyll chloroplast (Hatch and Slack 1967) (Fig. 1). Like phosphoenolpyruvate carboxylase and NADP-MDH, it is reversibly light activated (Slack 1968, Edwards et al. 1985), but through a unique phosphorylation/dephosphorylation cycle which involves a bifunctional regulatory protein, also responsible for its inactivation (Sugiyama 1974, Burnell and Hatch 1983, 1985). Low temperature or the absence of Mg 2+ or thiol compounds inactivates the enzyme (Shirahashi et al. 1978, Hatch 1979, Krall et al. 1989). Cold inactivation of the enzyme from sorghum, maize and Digitaria sanguinalis is accompanied by dissociation of the active tetrameric form into dimers (Shirahashi et al. 1978) or monomers (Hatch 1979). The absence of Mg 2+ results in the formation of dimers (Sugiyama 1973). Some variations were observed in chilling tolerance between C 4 species and cultivars (Sugiyama and Boku 1974, Sugiyama et al. 1979). Divalent cations such as Mn 2+ , Mg 2+ and Ca 2+ protect dikinase against inactivation at 0°C (Krall et al. 1989). The presence of physiological concentrations of P-enolpyruvate or pyruvate (Hatch, 1979, Shirahashi et al. 1978) or compatible solutes such as trimethylamine-N-oxide, glycinebetaine, glycerol and proline (to a lesser extent) offers an enhanced stability of PPDK at low temperature (Krall et al. 1989). Cold inactivation of enzymes has generally been related to dissociation (Bock and Frieden 1978). The loss of quaternary structure may be linked to a temperature-induced shift in the pKa

167 of certain groups involved in the maintenance of the oligomeric state and/or a decrease in hydrogen bond strength. In the case of PPDK, both factors seem to be involved with dissociation, in view of the dependence of dissociation on pH and the presence of organic cosolutes, which could decrease the capability of water to disrupt hydrogen bonding (Gerlsma 1978). Additionally, the greater stability of PPDK in binary solvents could be the result of an increased self-association of the protein, according to the exclusion volume theory (Gekko and Timasheff 1981a,b). As Krall et al. (1989) discuss in a recent paper, the oligomeric structure of PPDK could be affected in vivo by pH, protein and divalent cation concentrations and the level of organic solutes. It is not clear at present whether and how the phosphorylation-dephosphorylation processes accounting for enzyme regulation could influence its oligomeric state, especially considering that the dark and light enzyme-forms were reported as tetrameters (Sugiyama 1973, Sugiyama and Iwaki 1977).

PPDK regulatory protein PPDK is interconverted from an active nonphosphorylated form to an inactive threonylphosphorylated form by two different enzymatic reactions catalyzed by a single protein, named PPDK-regulatory protein (PPDK-RP) (Sugiyama 1974, Burnell and Hatch 1983, 1985) (Fig. 1). Both reactions are mechanistically unique, involving ADP as the phosphate donor for inactivation and Pi as the acceptor for the phosphorolytic activation (Burnell and Hatch 1983, 1985). The regulatory protein was first partially purified by Sugiyama (1974), and was reported to have a molecular weight of 90 kDa when purified (Nakamoto and Sugiyama 1982). Subsequently, Burnell and Hatch (1983) found that the molecular weight of the partially purified PPDK-RP was pH-dependent, eluting as a dimer of 90 kDa at pH 7.5 and as tetramer of 180 kDa at pH8.3 after size-exclusion chromatography. PPDK-RP is unstable in solution (Edwards et al. 1985), but its activity may be preserved by the addition of blue dextran, Pi, ATP, the organic

cosolute sorbitol (Sugiyama 1974, Burnell and Hatch 1985) or casein (Baer and Schrader 1985). The physiological regulation of PPDK-RP activity is not well understood in vivo (Usuda 1988, Roeske and Chollet 1989), although pyruvate has been shown to play a dominant role in vitro (Budde et al. 1986). Although the enzyme undergoes association-dissociation between pH 8.3 and 7.5, it is not established that these changes alter its catalytic properties (e.g., see Burnell and Hatch 1983). Moreover, the pH dependence of both the activation (dephosphorylation) and inactivation (phosphorylation) reactions have a broad optimum between pH 7.8 and 8.3 (Burnell and Hatch 1985), precluding, at first sight, oligomeric interconversion as a regulatory agent.

Final comment The regulation of C 4 metabolism has been the focus of attention of many plant biochemists and physiologists for several years. Much of the research has centered on the study of the kinetic and structural properties of the enzymes involved. More recently, the reversible redox and phosphorylation processes that alter their properties have been examined. Most of the C 4 enzymes are oligomeric proteins, and all of these have been demonstrated to undergo reversible polymerization under different in vitro conditions. The question as to how much this factor contributes to enzyme regulation in vivo is still unanswered. However, as has been outlined in this review, there are sufficient data to suggest that the quarternary structure of some of the C4-enzymes (most notably NADP- and NADME) may be implicated, at least in part, in the regulation of the pathway. Unequivocal evidence from future work is required to support this hypothesis, and it should be focused in such a way as to best simulate the in vivo conditions during the activity/molecular weight determinations.

Acknowledgements The authors' work was supported by grants from the Consejo Nacional de Investigaciones Cien-

168

tificas y T6cnicas (CONICET), Argentina. CSA and AAI are career members of CONICET. CSA is a recipient of a fellowship from the John Simon Guggenheim Memorial Foundation. We thank Dr Tatsuo Sugiyama, University of Nagoya, Japan, and Dr Raymond Chollet and Dr Marion H. O'Leary, University of NebraskaLincoln, USA, for critical reading of the manuscript and many helpful discussions.

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