The activity of the enzyme ribulose bisphosphate carboxylase (RuBPCase) was estimated after rapidly extracting it from intact wheat leaves pretreated under different light and CO2 levels. No HCO3− was added to the extraction buffer since it is shown to inhibit RuBPCase. The activity increased as light intensity or CO2 concentration during pretreatment was increased. Enzyme activity increased as temperature during pretreatment was decreased. Light activation did not affect the affinity of RuBPCase for CO2. A Km of 30 μM CO2 under air level O2 was determined. CO2, light and temperature are three main limiting factors of photosynthesis. It seems that the activity of RuBPCase is regulated by these factors according to the requirements for CO2 fixatio

MÄCHLER, F.

NÖSBERGER, J.

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Journal of Experimental Botany, Vol. 31, No. 125, pp. 1485-1491, December 1980Regulation of Ribulose Bisphosphate CarboxylaseActivity in Intact Wheat Leaves by Light, CO2, andTemperatureF. MACHLER AND J. NOSBERGERInstitutfur Pflanzenbau, ETH-Zurich, Universitatsstrasse 2, CH-8092 Zurich,SwitzerlandReceived 27 March 1980ABSTRACTThe activity of the enzyme ribulose bisphosphate carboxylase (RuBPCase) was estimated afterrapidly extracting it from intact wheat leaves pretreated under different light and CO2 levels. NoHCO7 was added to the extraction buffer since it is shown to inhibit RuBPCase. The activityincreased as light intensity or CO2 concentration during pretreatment was increased. Enzymeactivity increased as temperature during pretreatment was decreased. Light activation did notaffect the affinity of RuBPCase for CO2. A Km of 30 pM CO2 under air level O2 was determined.CO2, light and temperature are three main limiting factors of photosynthesis. It seems that theactivity of RuBPCase is regulated by these factors according to the requirements for CO2 fixation.I N T R O D U C T I O NRibulose-1,5-bisphosphate carboxylase (RuBPCase) is an enzyme of the photo-synthetic carbon reduction cycle and catalyses the reaction of CO2 with RuBP toform 3-phosphoglyceric acid. Purified enzyme from wheat leaves shows differentdegrees of activation depending on the concentrations of CO2 and Mg2+, pH, andtemperature. Kinetic analyses have indicated that the enzyme is activated in threesteps according to the equation E* *± E + CO2*± E-CO2 + Mg2+ J± E-CO2-Mg2+(Lorimer, Badger, and Andrews, 1976; Laing and Christeller, 1976; Chollet andAnderson, 1977; Machler, Keys, and Cornelius, 1980). The first step may be aconformational change of the protein and is influenced by temperature. Activationof RuBPCase can be shown not only in vitro but also in vivo. The degree ofactivation of enzyme in intact chloroplasts is shown to depend on CO2 and light(Bahr and Jensen, 1978). Part of the light activation is most likely due to increasedMg2+ and pH in the stroma (Werdan, Heldt, and Milovancev, 1975; Portis andHeldt, 1976). This paper reports experiments in which intact wheat leaves weretreated under different CO2 and light levels before RuBPCase was extracted rapidlyfor estimation of activity.1486 Mdchler and Nosberger—Regulation ofR uBP CarboxylaseM A T E R I A L S A N D M E T H O D SWheat (Triticum aestivum var. Kolibri) was grown in soil in cabinets at 20 °C/15 °C day/nighttemperatures and a light intensity of 300 //E m~2 s~' with a day length of 16 h. Seedlings 10 d oldwere pretreated in glass vessels with air of different CO2 concentrations and with different lightintensities. For preparation of gas mixtures Wosthoff pumps were used. The light source was a 400W sodium vapour lamp separated from the plants by a 6 cm water layer.For rapid extraction of enzyme the leaves of five seedlings were cut into pieces of about 2 cm inlength and homogenized in a mortar with 3 g sand and 4 ml 20 mM Tris-HCl (pH 8-2), 20 mMMgCl2, and 10 mM mercaptoethanol. The homogenate was diluted with 25 ml of the same solutionand filtered through a nylon net (20 fim mesh opening) by placing an open-ended rubber cylinderwith the net attached to the bottom end into the homogenate. 20 (A of the filtrate which rose upinside the cylinder were tested for carboxylase activity at 10 °C. Mortar, sand, and solutions wereprecooled in ice. The entire extraction procedure was completed in less than 1 min.To study the influence of pH and HCOj concentration on the extracted enzyme 4 g freshly cutleaves were homogenized with 6 g sand and 8 ml 20 mM Tris-HCl (pH 8-2), 20 mM MgCl^ and10 mM mercaptoethanol. The homogenate was filtered through two layers of muslin andcentrifuged at 40 000 g for 20 min at 4 °C. The extract was used within 2 h. 0-1 ml extract wasadded to 0-9 ml of either 500 mM Tris-HCl (pH 8-3) or 250 mM HEPES (pH 6-8) with 20 mMMgCl2, 10 mM mercaptoethanol, and different NaHCO3 concentrations in 50 ml Erlenmeyer flasks.The flasks were placed in a water bath of 20 °C and gassed with air of different CO2 content, whichwas in equilibrium with the HCO7 concentrations of the solutions (calculation of the concentrationsaccording to Umbreit, Burris, and Stauffer, 1972). After 10 min 10 /d solution were tested forcarboxylase activity at 25 °C.The enzyme activity was assayed by the procedure of Lorimer et al. (1977) using 0-5 ml 84 mMTris-HCl (pH 8-2), 10 mM mercaptoethanol, 0-4 mM RuBP, 20 mM MgCl2, and 20 mMNaHI4CO3 (1 Ci mol"1). For determination of the kinetic constants the reaction mixtures wereprepared under CO2-free air. The reaction was started by adding the enzyme and stopped afteT 30 sby adding 0-1 ml 2 N HC1. Of this mixture 0-4 ml was transferred to a scintillation vial and dried at90 °C. The residue was dissolved in 1 ml water, 10 ml scintillator solution (Patterson and Greene,1965) were added, and the amount of I4C present was measured using a scintillation spectrometer.Soluble protein was estimated according to Bensadoun and Weinstein (1976).RESULTS AND D I S C U S S I O NThe activation ofRuBPCase in intact leaves by light and CO2The purpose of the experiments was to examine the degree of activation ofRuBPCase in intact leaves. Therefore the amount of the active complexE-CO2-Mg2+ had to be kept as constant as possible during extraction. This wasachieved by adding Mg2+ in excess to the extraction medium in order to inhibit thedissociation of the active complex, and by keeping the CO2 concentration low inorder to avoid the activation of the inactive enzyme.The activity of RuBPCase, rapidly extracted from intact leaves, was dependentupon light intensity and CO2 concentration during preincubation. The activityincreased as CO2 concentration (Fig. 1) or light intensity was increased (Fig. 2).Temperature showed a marked effect on the activity of RuBPCase in intactleaves. Preincubation at 7 °C at a light intensity of 300 /uE m~2 s~! under air levelCO2 resulted in an activity of 0-13 /umo\ CO2 min""1 mg"1 protein as comparedwith 0-07 //mol CO2 min"1 mg"1 protein for preincubation at 25 °C. The assaytemperature was 10 °C. This is obviously not a direct effect of temperature on theprotein, since studies with purified enzyme show that RuBPCase activity decreasesas temperature is decreased (Machler et al. 1980). In intact leaves temperatureseems to affect enzyme activity by changing the level of regulatory metabolites.As has already been shown with protoplasts (Robinson, McNeil, and Walker,Mdchler and Nosberger—Regulation ofRuBP Carboxylase 14870.12ea.E 0.06"5£a.OO0 0.03 0.1 0.3 1 3 10Percentage CO2 in air during incubationFIG. 1. Influence of percentage CO2 in air during 2 h incubation of wheat seedlings in the dark atroom temperature on activity of rapidly extracted RuBPCase. The enzyme was tested at 10 °C. Fordetails see Materials and Methods.1979), dark incubation does not inactivate RuBPCase completely. Leavesincubated in the dark in 10% CO2 attained activities similar to leaves which hadbeen illuminated at 800 /iE m~2 s"1 under air level CO2. Enzyme in leaves incubatedin the dark in 10% CO2 was activated relatively slowly, requiring 1 h. This may bedue to very low levels of Mg2+ present in the stroma when it is dark (Portis andHeldt, 1976).0.12eo.•§0.063400 800Light intensity (^E m 2 s ') during incubationFio. 2. Influence of light intensity during 2 h incubation of wheat seedlings in air at roomtemperature on the activity of rapidly extracted RuBPCase. The activity of enzyme was tested at10 °C. For details see Materials and Methods.1488 Mdchler and Nosberger—Regulation ofRuBP Carboxylase0.09 rSQ.E 0.045•3Ou2 3 4 5Hours of light and dark during incubationFio. 3. Influence of light (100 fiE m~2 s~') and dark during incubation of wheat seedlings in air atroom temperature on the activity of rapidly extracted RuBPCase. The activity of enzyme was testedat 10 °C. For details see Materials and Methods.The induction of photosynthesis by illumination was accompanied by a rapidincrease of enzyme activity followed by a decline and levelling off (Fig. 3). Thedecline can be explained by a decrease in CO2 and an increase in RuBPconcentration, the latter being known to inhibit RuBPCase (Sicher and Jensen,1979; Laing and Christeller, 1976).In contrast to Bahr and Jensen (1978) no difference in affinity of RuBPCase forCO2 was found between light and dark treatments (Fig. 4). In both cases a Km of30 fjM CO2 under air level O2 was determined.The concentration of the substrate CO2 and the intensity of light as source ofenergy are two of the main limiting factors of photosynthesis. The activity ofRuBPCase seems to be regulated by the degree to which these factors are present.Temperature is another factor affecting CO2 fixation. The carboxylation of RuBPin vitro shows a sharp decrease with temperature (Machler et ah, 1980). In vivothis decrease can be partly compensated for by an increased solubility of thesubstrate CO2 at low temperatures and an increased activation of RuBPCase.The stability of enzyme activity during extraction in the absence and presence ofRuBPCase extracted from intact leaves in the absence of HCOj changed itsactivity only slowly within the next few minutes (Fig. 5). If 20 mM NaHCO3 waspresent in the extraction medium, the activity changed rapidly. Relatively inactiveenzyme from leaves incubated in CO2—free air was activated, whereas highly activeenzyme from leaves incubated in 10% CO2 was inactivated by NaHC03 . Fourminutes after extraction both enzymes had attained the same level of activityregardless of how the leaves had been preincubated. Extracted enzyme is known toMdchler and Nosberger—Regulation ofRuBP Carboxylase 1489(mM C02)Flo. 4. Double reciprocal plots of RuBPCase activity as a function of CO2 concentration with airlevel O, in the assay medium. RuBPCase was extracted rapidly from leaves incubated for 30 min inthe dark ( • ) or at a light intensity of 800 /& m~J s~' (O) both at room temperature and 300 parts10-* COj. The activity of enzyme was tested at 10 °C. For details see Material! and Methods.be activated by CO2, not by HCO7 (Lorimer et ah, 1976). Figure 5 suggests thatHCO7 inhibits the enzyme. This suggestion is supported by-the interaction of CO2and pH on the activity of extracted RuBPCase (Fig. 6). The combination of anincreased CO2 concentration with a high pH inactivates RuBPCase, probably due0.1 rci"oa.•ouKOo0.051 2 3 4 5Minutes since start of extractionFio. 5. Influence of presence (A, A) and absence (O, • ) of 20 mM NaHCO, in the extractionmedium on the activity of RuBPCase. The enzyme was extracted rapidly from wheat leavespreincubated for 2 h in CO2 free air (A, • ) or 10% CO2 (A, O) in the dark at room temperature.The enzyme was tested at 10 °C. For details see Materials and Methods.1490 Mdchler and Nosberger—Regulation ofRuBP Carboxylase0.8r ' .;pH 6.8oo.00EI§0.4oBopH8.30 0.03 0.1 0.3 1 3 10Percentage CO2 in air above the solutionFio. 6. Influence of CO2 concentration and pH (O:pH 6-8, #:pH 8-3) on the activity ofRuBPCase in crude extracts from wheat leaves. The CO2 concentrations in the extracts were inequilibrium with the indicated CO2 concentrations in the air above the extracts. The enzyme activitywas tested at 25 °C. For details see Materials and Methods.to the equilibrium with an increased concentration of HCO7. The high activity ofenzyme extracted from leaves which had been incubated in 10% CO2 (Fig. 5) isprobably due to a low concentration of HCO7 in the stroma, since the pH of thestroma must be decreased by high concentrations of carbonic acid.A comparison of the activity of rapidly extracted enzyme with purified enzymeactivated by CO2 andMg2+Vmnx of enzyme rapidly extracted from illuminated leaves was 0-12 //mol CO2min"1 mg~: protein at 10 °C. Assuming activity to be as similarly dependent ontemperature as purified enzyme (Machler et ai, 1980) and assuming 50%RuBPCase present in soluble protein, a specific activity of 1-8 /anol CO2min"1 mg~' protein at 25 °C was calculated. This value is high compared with theestimates of activated purified enzyme. Purified wheat RuBPCase activated by CO2and Mg2+ attained a specific activity of 0-75 /imol CO2 min"1 mg"1 protein at25 °C (Machler et at., 1980). Purified spinach extracts showed a specific activity of1-8 /umol CO2 min"1, mg"1 protein at 30 °C (Lorimer et ai, 1977). However it issuggested that purified enzyme fully activated by CO2 and Mg2+, but not inhibitedby HCO7, would attain a much higher specific activity.ACKNOWLEDGEMENTThe project was supported by the Swiss National Science Foundation. The authorsthank Mrs. Marcia Bocksch for skilful technical assistance.Mdchler and Nosberger—Regulation ofR uBP Carboxylase 1491LITERATURE CITEDBAHR, J. T., and JENSEN, R. G., 1978. Archs. Biochem. Biophys. 185,39-48.BENSADOUN, A., and WEINSTEIN, D., 1976. Analyt. Biochem. 70,241-50.CHOLLET, R., and ANDERSON, L. L., 1977. Biochim. biophys. Ada, 482,228-40.LAING, W. A., and CHRISTELLER, J. T., 1976. Biochem. J. 159,563-70.LORIMER, G. H., BADGER, M. R., and ANDREWS, T. J., 1976. Biochemistry, 15,529-36.1977. Analyt. Biochem. 78,66-75.MACHLER, F., KEYS, A. J., and CORNELIUS, M. J., 1980. J. exp. Bot. 31,7-14.PATTERSON, M. S., and GREENE, R. C, 1965. Analyt. Chem. 37,854-7.PORTIS, A. R., and HELDT, H. W., 1976. Biochim. biophys. Ada, 449,434^6.ROBINSON, S. P., MCNEIL, P. H., and WALKER, D. A., 1979. FEBS Lett. 97,296-300.SICHER, C. S., and JENSEN, R. G., 1979. PL Physiol., Lancaster, 64,880-83.UMBRErr, W. W., BURRIS, R. H., and STAUFFER, J. F., 1972. Manometric and biochemicaltechniques, 5th edn., Burgess Pub. Co., Minneapolis.WERDAN, K., HELDT, H. W., and MILOVANCEV, M., 1915.Biochim. biophys. Ada, 396,276-92.

Regulation of Ribulose Bisphosphate Carboxylase Activity in Intact Wheat Leaves by Light, CO2, and Temperature

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