Glycine, sarcosine, and TMAO weremore effective than betaine or KCI in lowering the extent of the dissociation of the enzyme molecule. Organisms that thrive in ...
Plant Physiol. (1986) 81, 1044-1049 0032-0889/86/81/1044/06/$0 1.00/0
Effect of Betaine on Enzyme Activity and Subunit Interaction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Aphanothece halophytical Received for publication February 4, 1986 and in revised form April 30, 1986
ARAN INCHAROENSAKDI*2, TETSUKO TAKABE, AND TAKASHI AKAZAWA
Research Institute for Biochemical Regulation, School ofAgriculture, Nagoya University, Chikusa, Nagoya 464, Japan ABSTRACT The presence of betaine, a quaternary ammonium compound, at a concentration (0.5 molar) reported to accumulate inside Aphanothece halophytica in response to increasing external salinity, slightly promoted ribulose-1,5-bisphosphate (RuBP) carboxylase activity. KCI at 0.25 molar inhibited RuBP carboxylase about 55%. Betaine relieved the inhibition by 0.25 M KCI and the original uninhibited activity was restored at 1 M betaine. Other osmoregulatory solutes such as sucrose and glycerol also reduced KCI inhibition, though to a lesser extent than betaine. Proline had no effect. The protective effect of betaine against KCI inhibition of RuBP carboxylase activity was also observed in other cyanobacteria, i.e. Synechococcus ACMM 323, Plectonema boryanum, and Anabaena variabilis, and in the photosynthetic bacterium Rhodospirillum rubrum but not in Chromatium vinosum. Apart from betaine, other quaternary ammonium compounds, i.e. sarcosine and trimethylamine-N-oxide (TMAO), but not glycine, also protected the enzyme against KCI inhibition and the effectiveness of such compounds appeared to correlate with the extent of N-methylation. Heat and cold inactivation of the enzyme could be protected by either betaine or KCI. However, best protection occurred when both betaine and KCI were present together. The Km (CO2) was not altered by either betaine or KCI, nor when they were present together. However, the Km (RuBP) was increased about 5-fold by KCI, but was unaffected by betaine. The presence of betaine together with KCI lowered the KCI-raised Km (RuBP) by about half. The extent of the dissociation of the enzyme molecule under the condition of low ionic strength was reduced by either betaine or KCI alone and more so when they were present together. Glycine, sarcosine, and TMAO were more effective than betaine or KCI in lowering the extent of the dissociation of the enzyme molecule.
Organisms that thrive in hypersaline environments must possess specific mechanisms to adjust their internal osmotic status according to the salinity of the environment. One such mechanism is the ability to accumulate inorganic ions such as K+, and Na+ to a lesser extent, or some organic solutes like glycerol, ' Supported by research grants from the Ministry of Education, Science and Culture (Mombusho) of Japan (60560089) to T. T. and the Nihon Seimei Zaidan (Tokyo) to T. A. and T. T. This is Paper No. 68 in the series "Structure and Function of Chloroplast Proteins." 2 Recipient of the scholarship from the Japanese Government (Mombusho). Permanent address: Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, Thailand.
sucrose, proline, and betaine (11). Osmoregulation in cyanobacteria grown at high external NaCl concentrations involves the accumulation of organic and inorganic solutes. For several fresh water cyanobacteria the role of low mol wt carbohydrates as osmoregulators has been shown; for example, glucosylglycerol in Synechocystis 6714 (14) and sucrose in Synechococcus 6311 (2). A combination of both inorganic and organic solutes (sucrose and glucosylglycerol) is involved in osmotic adjustment in the euryhaline cyanobacter-
ium, Synechocystis PCC 6714, which can grow in both nonsaline and saline media (19). Betaine (glycinebetaine), a quaternary ammonium compound, was first shown to be the major osmoticum in a halophilic
cyanobacterium, Synechocystis DUN 52 (16). Aphanothece halophytica is a halophilic cyanobacterium, unable to grow at or below 3.5% (w/v) NaCl (sea water salinity) (5). The osmotic adaptation of this organism was first studied by Miller et al. (15) who showed that A. halophytica accumulated up to 1 M K+ in response to increasing external salinity. Recently, Reed et al. (18) demonstrated that betaine is the major solute accumulated followed by K+ in four strains of cyanobacteria including A. halophytica grown in high salinity. Furthermore, the variation for betaine accumulation in response to changes in extracellular salinity is much greater than that observed with K+. The former group has also recently accepted the idea that betaine is a major osmoticum in A. halophytica cells (21). Betaine has also been implicated as a major cytoplasmic osmoticum in shoots of a number of plants (25) as well as in the halophilic photosynthetic bacterium, Ectothiorhodospira halochloris (7). High concentration of salts have been reported to inhibit the activity of many enzymes from both eucaryotic and procaryotic origins (28). Higher plants are able to compartmentalize the accumulated salts, Na+ and Cl- in particular, in the vacuole (8, 26), and thereby prevent the inhibition of enzyme activities. In contrast, the soluble enzymes in procaryotic organisms have to be directly exposed to any osmoregulatory substances. Based on the findings of Reed et al. (18) it is interesting to investigate how salt and betaine may affect the function of some soluble enzymes in A. halophytica. We reported previously that KCI strongly inhibits the activity but prevents the dissociation of RuBisCO3 molecules from A. halophytica into catalytic core (octamer of large subunit A) and small subunit B (27). In the present study, we examined the effect of betaine and some other solutes in relation to the effect of KCI on the activity of RuBP carboxylase, a key enzyme in Abbreviations: RuBisCO, ribulose-l1,5-bisphosphate carboxylase/oxygenase; PEG, polyethyleneglycol; RuBP, ribulose-1 ,5-bisphosphate; TMAO, trimethylamine-N-oxide.
1044
I
BETAINE AND RuBisCO
photosynthesis, from A. halophytica. Effects of betaine and KCI heat and cold inactivation of RuBP carboxylase were also investigated. We demonstrated previously that RuBisCO molecules from A. halophytica can be partially dissociated under conditions of low ionic strength and low temperature (12). The protective effect of quaternary ammonium compounds on the dissociation of RuBisCO molecule at low temperature from A. halophytica is also reported. on
MATERIALS AND METHODS Organisms and Growth Conditions. Aphanothece halophytica was grown autotrophically as previously described (27). Plectonema boryanum and Anabaena variabilis were grown in a BG 11 medium (23). The culture and growth of Chromatium vinosum have been described elsewhere (13). Purification of RuBisCO. RuBisCO from A. halophytica was purified by sucrose density gradient centrifugation as previously described (12) with slight modification. The 25 to 50% (NH4)2S04 pellet, after resuspension in 50 mM Hepes-KOH buffer (pH 7.5) containing 1 mM EDTA, 5 mM DTT, and 0.3 M KCI (HEDK buffer) and passing through a Sephadex G-25 column equilibrated with the same buffer (referred to as partially purified enzyme), was layered onto a 38 ml 0.2 to 0.8 M linear sucrose gradient prepared in HEDK buffer containing 10 mM CaCl2 and 20 mM MgCl2. The gradient was centrifuged in a Beckman VTi 50 rotor at 242,000g for 2 h at 40C in a Beckman ultracentrifuge. The enzymically active fractions were pooled and precipitated with 60% (NH4)2SO4. The resuspended pellet finally passed through a Sephadex G-25 column to remove low mol wt compounds. Partially purified enzymes from P. boryanum and A. variabilis were obtained by the method described for A. halophytica with the omission of KCI in the purification buffer. RuBisCO from C. vinosum was partially purified by a modification of the method of Berhow and McFadden (1). About 10 g of cells were suspended in 50 ml of MEMMB buffer (50 mM morpholinopropanesulfonic acid, 0.1 mM EDTA, 1 mM 2-mercaptoethanol, 10 mM MgCl2, 50 mM NaHCO3, adjusted to pH 7.2 at 250C) and sonicated twice, 5 min each at 200 W in a Kubota Insonator model 200 M. The resulting suspension was centrifuged at 94,000g for 1 h. The supematant was diluted with two volumes of cold MEMMB buffer and was adjusted to 10% (w/v) PEG 4,000 by slow addition of 60% PEG. The solution was stirred in an ice-bath for 30 min before centrifugation at 39,000g for 20 min to remove most of the chromatophores and membranous fractions. Additional protein was precipitated from the supernatant by adding 2 M MgCl2 to a final concentration of 50 mm. The solution was stirred in an ice-bath for 30 min before centrifugation as before. The final pellet obtained was resus-
1045
RESULTS Effect of Betaine and Other Solutes on RuBP Carboxylase Activity from A. halophytica. A range of organic solutes that have been implicated as cytoplasmic osmoregulators in plants or photosynthetic procaryotes were examined in comparison to KCI for their effects on the activity of RuBP carboxylase from A. halophytica (Fig. IA). Sucrose enhanced enzyme activity even at concentrations up to 1 M. The effect of betaine was similar to that of glycerol, which showed a slight activation of activity. On the other hand, proline and KCl had an inhibitory effect on enzyme activity. Fifty percent of the control activity was observed at 0.25 M KCI, confirming our previous results (27). For betaine and glycerol, our results are in agreement with previous studies that showed slight activation of malate dehydrogenase from Suaeda maritima plants by betaine (6) and slight activation by glycerol of glucose 6-P dehydrogenase from the unicellular alga, Dunaliella (3). However, striking differences from previous studies were observed with sucrose and proline. Sucrose was slightly (17) or strongly (29) inhibitory to leaf malate dehydrogenase, 140
0120
P~~~~~~~~Gtrole-
_ 60 _ X
was
any
a small volume of 10 mm phosphate buffer (pH 7.6) containing I mM EDTA and passed through a Sephadex G-25 column eluted with the same buffer before use. Assay Method of RuBP Carboxylase and Protein. The standard assay mixture for RuBP carboxylase contained the following components in a total volume of 250 ,ul: 100 mm Tris-HCI (pH
0
40 20 0
0 175
pended in
7.8), 20 mm MgCl2, 50 mm Na'4HCO3 (0.4 mCi/mmol), 1 mM RuBP, and appropriate amounts of purified enzyme. After a 10 min activation at 250C, the catalytic reaction was initiated by the addition of RuBP. The reaction was stopped after 10 min by the addition of 50 Ai of acetic acid. The vial contents were evaporated to dryness under an electric lamp, 0.3 ml of water, and 3 ml of scintillation fluid were added, and the acid-stable radioactivity was determined by liquid scintillation counting. Protein was assayed by using the Bio-Rad protein assay kit and Sigma
BSA
as a
standard.
t
0
0.25
0.5
0.75
1.0
Sokite (M) FIG. 1. Comparative effects of various solutes on RuBP carboxylase activity from A. halophytica. A, Five gl of partially purified enzyme (8.7 gg protein) was assayed in a total volume of 250 ul containing 25 mM Tris-HCI (pH 7.8), 20 mm MgCI2, 10 mM NaH'4CO3 (0.4 mCi/mmol), 1 mm RuBP, and various concentrations of solutes as indicated (each solute was prepared in distilled H20 and the pH was adjusted to 7.8 before use). Other experimental details are described in the text. B, the same experimental protocol as (A) was employed except that 0.25 M KCI was present in all assays.
INCHAROENSAKDI ET AL.
1046
whereas proline either had no effect (24) or slightly activated (6) some enzymes from halophytes. The investigation of the protective effects of betaine and other solutes on inhibition of RuBP carboxylase by KCl revealed that all but proline were effective (Fig. 1 B). Betaine was most effective, especially at high concentrations followed by sucrose and glycerol. Proline did not appear to confer any protection but it is interesting to note that the presence of proline, even at high concentrations, did not further decrease the activity of KC1inhibited RuBP carboxylase (cf Fig. LA). Effect of Betaine Alone and Together with KCI on RuBP Carboxylase Activity in other Cyanobacteria and Photosynthetic Bacteria. The effect of betaine alone and together with KCI on RuBisCO activity was also studied in five other organisms: three cyanobacteria, Synechococcus ACMM323, Plectonema boryanum, Anabaena variabilis and two photosynthetic bacteria, Chromatium vinosum and Rhodospirillum rubrum. Figure 2 compares the responses of RuBP carboxylase activities from the various organisms to increasing betaine concentrations in the presence and absence of KCL. In the absence of KCl, the Synechococcus enzyme showed no response to betaine, whereas some activation at intermediate betaine concentrations was observed for the remaining enzymes with the exception ofthe Chromatium enzyme, which was inhibited 50% by 1 M betaine. Inhibition of enzyme activity by 0.25 M KCl was greatly relieved by increasing betaine concentrations in all cases but Chromatium, which again was inhibited by betaine. The protection afforded by betaine appeared to be most effective with the Aphanothece enzyme, especially at high betaine concentrations. It is noted that none of the organisms tested in Figure 2, except A. halophytica, have been reported to accumulate betaine. Obviously the stimulation of RuBisCO and the protection against KCI inhibition by betaine is not species specific. Influence of Solute Structure on Protection against KCI Inhibition of RuBP Carboxylase Activity from A. halophytica The effect of varying the N-methylation of glycine on the protection against KCl inhibition is shown in Figure 3. Glycine, with no
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Plant Physiol. Vol. 81, 1986
methyl group, was unable to relieve the inhibition by KC1. TMAO was more effective than betaine and sarcosine, and restored the original uninhibited activity at concentrations below 0.25 M.The relative effectiveness of the solute was correlated with the extent of methylation of the solute's nitrogen atom. These data are in agreement with previous studies on protection against urea inhibition of rabbit phosphofructokinase (10) and against KCI inhibition of barley malate dehydrogenase (6). Effect of Betaine and KCI on Heat and Cold Inactivation of RuBP Carboxylase Activity from A. halophytica. RuBisCO was heated at 50°C or cooled at 0°C in the presence and absence of betaine and/or KCI for various times prior to enzyme activity measurement at 25°C. The enzyme lost about half ofthe original activity after heating for 1 min in the absence of effectors (Fig. 4A). Betaine conferred slight protection against heat inactivation, whereas better protection was afforded by KCL. The best protection was observed when the enzyme was heated in the presence of betaine plus KCL. Similar responses of the enzyme to the effectors were observed during cold inactivation (Fig. 4B). However, betaine and KCl appeared to protect equally the enzyme against cold inactivation, in contrast to the better protective effect of KCl than betaine against heat inactivation. Effect of Betaine and KCI on Kinetic Properties of RuBP Carboxylase from A. halophytica. Apparent Michaelis constants and maximum velocity for both substrates of RuBP carboxylase from A. halophytica were determined in the presence and absence of betaine and KCl (Table I). Betaine and KCl increased and decreased Vmax respectively, to 1.32 and 0.72 Amol CO2 fixed. minm'.mg-'. The reduced Vmdav caused by KC1 was reversed by the further addition of betaine. The Km (C02) appeared to be unaltered in the presence of betaine or KCl or both. Betaine did not cause a significant change in Km (RuBP). KCl, however, increased the Km (RuBP) about 5-fold and the Km (RuBP) in the presence of KCl was lowered about half by the further addition of betaine. Protective Effect of Quaternary Ammonium Compounds on the Dissociation of RuBisCO Molecule from A. halophytica.
FIG. 2. Effect of betaine on activity of RuBP carboxylase from six different organisms assayed in the presence and absence of KCL. About 8 to 15 ,g protein of partially purified enzymes (A, C, D, F) and 1 to 2 Mg protein of purified enzymes from R. rubrum (E) and Synechococcus ACMM 323 (B) were assayed in the presence (_) and absence (ca) of 0.25 M KC1 at the indicated betaine concentrations exactly as described for Figure 1. Control activity is in the absence of betaine. A, A. haloph.vtica (data are derived from Fig. 1); B, Synechococcus ACMM 323; C, P. boryanum; D, A. variabilis; E, R. rubrum; F, C. vinosum.
BETAINE AND RuBisCO
100
it 75
50
25
Glycine
0I 0.75 0 0.25 1.0 0.5 Sokdte (M) FIG. 3. Protection of RuBP carboxylase activity from A. halophytica against KCI inhibition by derivatives of methylamines having different degrees of N-methylation. The assay was as described for Figure 1 with the indicated concentrations of glycine (A), sarcosine (A), betaine (0), and TMAO (0).
Previously we reported that KCI and the substrate RuBP reduced the dissociation of RuBisCO molecule from A. halophytica (1 2).The strong protective effect ofbetaine against KCI inhibition of RuBP carboxylase (Fig. 1B) prompted us to test the effect of betaine on the dissociation of this enzyme. The partially purified RuBisCO was layered onto sucrose gradients containing various solutes. As shown in Figure 5, A to D, higher enzyme activity was detected around fraction 12 in the gradient containing betaine than that without betaine (cf dark shaded area of Fig. 5, A and B). This higher enzyme activity was attributed to less dissociation of small subunit from the holoenzyme, because when a fixed amount of catalytic core (highly depleted of small subunit) was added to the top fractions (fractions 1-11) the recovered activity was less in the gradient containing betaine than that without betaine (cf light shaded area of Fig. 5, A and B). In this assay, recovered activity was a linear function of the concentration of small subunit (12). The observed shift of enzyme activity peak (fraction 12) from the protein peak (fraction
1=
15) results from the partial dissociation of the small subunit in the sucrose gradient since both catalytic core and small subunit are essential for enzyme activity. It is evident that the protective effect of betaine on the dissociation of the enzyme was not as great as that observed with KCI (Fig. SC). Interestingly, since the protective effect by KCI was saturated at concentrations higher than 0.3 M (A Incharoensakdi, T Takabe, T Akazawa, unpublished data), the protective effect appeared to be additive when betaine was added together with KCI in the gradient (Fig. 5D). The results may suggest that KCI and betaine reduce the extent of the dissociation of the enzyme by different mechanisms. To examine whether the hydrophobicity of the solute molecule has any effect on the dissociation of the enzyme subunits, solutes having different methyl groups on the nitrogen atom were tested for their effects on the dissociation of the enzyme. Glycine, sarcosine and TMAO all showed similar effectiveness in reducing the extent of the dissociation of RuBisCO molecule (Fig. 5, EG), judging by the same peak area under the curve around the catalytic core (fraction 12) together with the same amount of dissociated small subunit (light shaded area of Fig. 5, E-G). The effectiveness of the methylamine solutes in reducing the extent of the dissociation of the enzyme did not appear to correlate with the number of methyl groups on the molecule, since glycine was even more effective than betaine (cf. Fig. 5, B and E), suggesting the involvement of complicated mechanisms due to the structure of each quaternary ammonium compound.
DISCUSSION The results obtained in this study suggest that betaine acts as a useful osmoticum inside A. halophytica cells. The fact that betaine is not inhibitory to RuBisCO activity of A. halophytica also points to the characteristic compatibility of this solute. According to Reed et al. (18), K+ concentrations inside A. halophytica are in the range of 180 to 280 mM, depending on the external salinity, whereas the concentration of betaine is between 300 and 1000 mm. Our results, employing 0.25 M KCI in the assay, clearly demonstrate that the inhibitory effect of KCI on RuBP carboxylase can be overcome by betaine and that the higher the concentration of betaine, the greater is the effect observed. The value of protection by betaine against salt inhibition, mostly NaCl, of some enzymes as reported in higher plants (17) has not been critically evaluated when compared with A. halophytica because of the evidence that Na+ and Cl- ions are compartmentalized in the vacuole (8, 26) and the fact that salt levels in the cytoplasm and chloroplast have not yet been clearly
1001
FIG. 4. Protective effect of betaine and/or KCI against heat and cold inactivation of RuBP carboxylase activity.
1-
0 0
A, RuBisCO from A. halophytica (34 ,g) was preequi-
80
0
e
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20 O_
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Time (min)
librated at 25°C for 30 min in a total volume of 100 ul in 50 mM Tris-HCI (pH 7.8) buffer containing no effector (0); 0.5 M betaine (0); 0.3 M KCI (A); 0.5 M betaine plus 0.3 M KCI (A). The preequilibrated enzyme solution was then incubated in a 50C shaking water bath. At various time intervals, 12.5 Ml was removed and assayed for activity as described in the "Materials and Methods." B, The experiments were as described in (A) except that the preequilibrated enzyme solution was incubated at 0°C for the indicated time intervals.
1048
INCHAROENSAKDI ET AL.
Table I. Apparent Km and VmaA Values.fbr RuBP?CarboxYlasefrom A. halophYtica Determined uinder Various Conditions The purified enzyme was used and the basic experimental procedures were as described in the text except that the reaction was stopped after 1 min. For the determination of Km (CO2). a fixed 2.5 mm RuBP and varying NaHCO3 concentrations (5-50 mM) were employed, whereas for Km (RuBP), 50 mm NaHCO3 and 10 to 400 juM RuBP were employed. Since activity of the enzyme was sensitive to changes in ionic strength. the difference caused by varying NaHCO3 was corrected for by an equivalent amount of NaCl. This was not necessary for the case of RuBP due to the low concentrations of RuBP employed. Km and Vmn, (determined when NaHCO3 was the variable substrate) together with SE were determined by the statistical method of Wilkinson (30). CO2 concentrations were calculated using a pK value of 6.3 for the C02-HC03interconversion. Km (CO2) Km (RuBP) Addition Vr a, ,unol/min mg mM 92± 13 0.99 ± 0.15 1.17 ± 0.18 None 0.72 ± 0.20 1.29 ± 0.22 435 ± 55 0.25 M KCI 1.32 ± 0.31 70± 12 1.12 ± 0.16 0.5 M betaine 0.96 ± 0.26 1.32 ± 0.22 195 ± 32 0.25 M KCI plus 0.5 M betaine
0.2 ^
Plant Physiol. Vol. 81, 1986
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determined. Other possible compatible solutes, e.g. proline, glycerol, and sucrose, which accumulate in other organisms (11) have, with the exception of proline, somewhat similar effects to betaine in regard to the protective effect against salt inhibition of RuBisCO from A. halophytica. However, glycerol and sucrose were less effective than betaine and this may make them unlikely osmotica in this halophilic cyanobacterium. In fact, it has been reported that the carbohydrate content of A. halophytica in response to salinity changes accounts for only about 1% of the betaine content ( 18). The presence of both betaine and KCI proved to be efficient in protecting RuBisCO from A. halophvtica against heat and cold inactivation (Fig. 4). This phenomenon may be of some importance in terms of the adaptive mechanism employed in vivo by A. halophvtica so that conformational changes of the enzyme caused by extreme heat or cold are reduced, thus minimizing the activity loss. The study of the effect of betaine on the kinetic properties and on the dissociation behavior of RuBisCO was intended to delineate the molecular interaction of betaine with the enzyme. The results showed no alteration of the Kmn for either substrate (CO2 and RuBP) which suggests that betaine does not directly bind to active sites on the enzyme. The observation that KCI increased the K,,, (RuBP) (Table I) is in general agreement with other enzymes from marine invertebrates as well as from mammal (4, 9). The reduced V,7,,, in the presence of KCI appears to be caused by an anion (Cl-) rather than a cation (K+) since the substitution of CH3COO- for Cl- caused little or no inhibition of enzyme activity and that potassium salts with different monovalent anions (Cl-, Br-, NO3 ) at the same concentration showed different degrees of inhibition of enzyme activity (data not shown). Therefore, one of the factors contributing to the inhibition of RuBP carboxylase by salts is the competition of anions with RuBP. Since high contents of K+ and Na+ are present inside A. halophvtica (18), comparable anion contents should also be present to balance the charge difference. High concentration of anions, especially Cl- (about 100 mM), has been reported to accumulate in the chloroplasts of the halophyte Suaeda australis (20) and spinach (Table 1 of Ref. 20). The two main features of the betaine molecule are its dipole character at physiological pH values, and the crowding of three methyl groups at the positively charged end of the molecule. Betaine may help prevent the dissociation of the subunits (Fig. SB), by virtue of its zwitterion characteristic, by binding to charged groups in the large and small subunits of the enzyme.
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Fraction Number FIG. 5. Effect of various solutes on the dissociation of RuBisCO molecule from A. halophytica during sucrose gradient centrifugation. Partially purified RuBisCO derived from about 1 g cell was layered on top of a linear sucrose gradient (0.2-0.8 M, 38 ml) and centrifuged in a Beckman VTi 50 rotor for 2 h at 242,000g and 4°C. The gradient solution was prepared in (A), 50 mM Hepes-NaOH (pH 8.0), 1 mM EDTA, 5 mM DTT; (B), same as (A) plus 0.5 M betaine; (C), same as (A) plus 0.3 M KCI; (D), same as (C) plus 0.5 M betaine; (E), same as (A) plus 0.5 M glycine; (F), same as (A) plus 0.5 M sarcosine; (G). same as (A) plus 0.5 M TMAO. All the gradient solutions were adjusted to pH 7.8 at 25°C. After centrifugation, 1.5 ml fractions were collected and 25 ,ul aliquots were used for the assay of activity as described in the text (0). Separately. 25 ,ul aliquots of fraction 14 in (A) were added to aliquots (10 ml) of the upper fractions (fractions 1- 11) in experiments (A) to (G) and enzyme activities were assayed to estimate the magnitude of the dissociation of small subunit (0). The values in the figure represent activities after subtracting the residual activity of that without added fraction 14 and that of fraction 14 itself. The refractive index (A) patterns were identical in all seven gradients. The dark shaded peak represents the remaining activity of the RuBisCO molecule which reflects the extent of the dissociation of the enzyme. The lightly shaded peak represents the magnitude of the dissociated small subunit since the activity recovered after supplementation of the catalytic core is proportional to the concentration of small subunit (12).
BETAINE AND RuBisCO
Although the nature of the subunit binding domain has been suggested to be hydrophobic, as exemplified by the effectiveness of salt and high temperature in reducing the extent of the dissociation of the subunits (12), the ease of dissociation of the enzyme molecule bears no relation to the degree of methylation of the compound (Fig. 5). For the protective effects afforded by betaine against KCl inhibition of RuBP carboxylase, the correlation exists between the extent of protection and the degree of methylation (Fig. 3). This might be explained in terms of the energy and volume changes occurring during catalytic conformational changes as suggested by Somero et al. (22). KCI, a salting-in salt, promotes protein-water interaction with a concomitant increase in activation volume accompanied by a reduced maximum velocity. The more hydrophobic group, e.g. the three methyl groups on trimethylamine-N-oxide and betaine, can act at the protein-water surface resulting in a decrease of bound water at the surface, and hence, a decrease in activation energy during catalysis. However, the influence of concentrated salts on proteins and other macromolecules may involve extremely complex physical chemistry among salts, macromolecules, and water. In conclusion, the overall results indicate that the accumulation of high concentrations of both betaine and KC1 in A. halophytica has obvious advantages with respect to RuBisCO.
KCI is more effective than betaine in keeping the two-subunit structure of the enzyme but the drawback is its inhibition on the
enzyme activity. This, however, is offset by betaine, which serves compatible solute, thus maintaining the internal osmotic balance of the organism. The combined effect of both betaine and KCI in protecting the enzyme against heat and cold inactivation is another example of the advantage of the simultaneous accumulation of these two solutes. Acknowledgments-The authors thank Dr. G. A. Codd for helpful discussions as a
and Dr. S. C. Huber for critically reading the manuscript and some suggestions.
1.
2. 3. 4.
5. 6.
7.
LITERATURE CITED BERHOW MA, BA McFADDEN 1983 A rapid and novel method for purification of ribulose- 1 ,5-bisphosphate carboxylase from Chromatium vinosum. FEMS Microbiol Lett 17: 269-272 BLUMWALD E, RF MEHLHORN, L PACKER 1983 Studies of osmoregulation in salt adaptation of cyanobacteria with ESR spin-probe techniques. Proc NatI Acad Sci USA 80: 2599-2602 BOROWITZKA U, AD BROWN 1974 The salt relations of marine and halophilic species of the unicellular green algae, Dunaliella. Arch Microbiol 96: 37-52 BOWLUS RD, GN SOMERO 1979 Solute compatibility with enzyme structure and function: rationales for the selection of osmotic agents and end-products of anaerobic metabolism in marine invertebrates. J Exp Zool 208: 137-152 BROCK TD 1976 Halophilic-blue-green algae. Arch Microbiol 107: 109-111 FLOWERS TJ, JL HALL, ME WARD 1978 Salt tolerance in the halophyte, Suaeda maritima (L) Dum.: Properties of malic enzyme and PEP carboxylase. Ann Bot 42: 1065-1074 GALINSKI EA, HG TRUPER 1982 Betaine, a compatible solute in the extremely halophilic phototrophic bacterium Ectothiorhodospira halochloris. FEMS Microbiol Lett 13: 357-360
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8. GORHAM J, RG WYN JONES 1983 Solute distribution in Suaeda maritima. Planta 157: 344-349 9. GREANY GS, GN SOMERO 1980 Contributions of binding and catalytic rate constants to evolutionary modifications in Km of NADPH for muscle-type (M4) lactate dehydrogenase. J Comp Physiol 137: 115-121 10. HAND SC, GN SOMERO 1982 Urea and methylamine effects on rabbit muscle phosphofructokinase. J Biol Chem 257: 734-741 1 1. HELLEBUST JA 1976 Osmoregulation. Annu Rev Plant Physiol 27: 485-505 12. INCHAROENSAKDI A, T TAKABE, T AKAZAWA 1985 Factors affecting the dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase/ oxygenase from Aphanothece halophytica. Arch Biochem Biophys 237: 445453 13. KOBAYASHI H, T AKAZAWA 1982 Biosynthetic mechanism of ribulose-1,5bisphosphate carboxylase/oxygenase in the purple photosynthetic bacterium, Chromatium visnosum: 1. Inducible formation. Arch Biochem Biophys 214: 531-539 14. MACKAY MA, RS NORTON, U BOROWITZKA 1983 Marine blue-green algae have a unique osmoregulatory system. Mar Biol 73: 301-307 15. MILLER DM, JH JONES, JH Yopp, DR TINDALL, WE SCHMID 1976 Ion metabolism in a halophilic blue-green alga, Aphanothece halophytica. Arch Microbiol II1: 145-149 16. MOHAMMAD FAA, RH REED, WDP STEWART 1983 The halophilic cyanobacterium Synechocystis DUN 52 and its osmotic responses. FEMS Microbiol Lett 16: 287-290 17. POLLARD A, RG WYN JONES 1979 Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 144: 291-298 18. REED RH, JA CHUDEK, R FOSTER, WDP STEWART 1984 Osmotic adjustment in cyanobacteria from hypersaline environments. Arch Microbiol 138: 333337 19. REED RH, SRC WARR, DL RICHARDSON, DJ MOORE, WDP STEWART 1984 Multiphasic osmotic adjustment in a euryhaline cyanobacterium. FEMS Microbiol Lett 28: 225-229 20. ROBINSON SP, WJ DOWNTON 1985 Potassium, sodium and chloride ion concentrations in leaves and isolated chloroplasts of halophyte Suaeda australis R.Br. Aust J Plant Physiol 12: 471-479 21. SIBLEY MH, JH Yopp 1985 Occurrence and osmoregulatory role of S-adenosylhomocysteine hydrolase in betaine synthesis by the halotolerant cyanobacterium, Aphanothece halophytica. Plant Physiol 77: S-141 22. SOMERO GN, M NEUBAUER, PS Low 1977 Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: evidence for enzyme hydration changes during catalysis. Arch Biochem Biophys 181: 438-446 23. STANIER RY, R KUNISAWA, M MANDEL, G COHEN-BAZIRE 1971 Purification and properties ofunicellular blue-green-algae (order chroococcales). Bacteriol Rev 35: 171-205 24. STEWART GR, JA LEE 1974 The role of proline accumulation in halophytes. Planta 120: 279-289 25. STOREY R, RG WYN JONES 1977 Quaternary ammonium compounds in plants in relation to salt resistance. Phytochemistry 16: 447-453 26. STOREY R, MG PITMAN, R STELZER 1983 X-ray micro-analysis of cells and cell compartments ofAtriplex spongiosa. J Exp Bot 34: 1196-1206 27. TAKABE T, AK RAI, T AKAZAWA 1984 Interaction of constituent subunits in ribulose-1,5-bisphosphate carboxylase from Aphanothece halophytica. Arch Biochem Biophys 229: 202-21 1 28. VON HIPPEL PH, T SCHLEIC 1969 The effects of neutral salts on the structure and conformational stability of macromolecules in solution. In SN Timasheff, ed, Structure and Stability of Biological Macromolecules, Vol 2. Marcel Dekkar, New York, pp 417-574 29. VON WILLERT DJ 1974 Effect of sodium chloride on respiration and the activity of malate dehydrogenase in some halophytes and glycophytes. Oecologia 14: 127-137 30. WILKINSON GN 1961 Statistical estimations in enzyme kinetics. Biochem J 80: 324-332
