Association of Carbonic Anhydrase Activity with - Plant Physiology

Plant Physiol. (1992) 100, 784-793 0032-0889/92/100/0784/1 0/$01 .00/0

Received for publication March 2, 1992 Accepted May 29, 1992

Association of Carbonic Anhydrase Activity with Carboxysomes Isolated from the Cyanobacterium Synechococcus PCC7942' G. Dean Price*, John R. Coleman, and Murray R. Badger Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, 2601, Australia (G.D.P., M.R.B.); and Department of Botany, University of Toronto, 25 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 (I.R.C.) ABSTRACT

for C02, conferring on the cell an increase in photosynthetic performance. The two major components of the CCM so far identified are the C1 transport system and the Rubisco-containing carboxysomes. The transport system comprises a constitutively expressed, active transporter for the C1 species C02 and HC03-, allowing C1 to be accumulated inside the cell as HC03- (18, 20, 28). Carboxysomes are small polyhedral bodies that are bounded by a thin protein shell (7) and that contain the majority of the cell's Rubisco activity. Carboxysomes appear to act as microcompartments where the conversion of HC03- to C02 is catalyzed by a low level of carbonic anhydrase (18, 23, 24). Some property of the carboxysome, possibly the protein shell around the structure, acts to impede the leakage of C02 away from the site of carboxylation, allowing elevated C02 levels to be maintained around Rubisco (18, 19). Through the controlled expression of human CA in the cytosol of Synechococcus PCC7942, it has been possible to show that Ci species in the cytosol are in slow equilibrium and that HC03- is the species delivered inside the cell by the C1 pump (18). Consequently, and through the use of various theoretical models (23, 24) and the analysis of various high C02-requiring mutants (19), it has become apparent that CA activity must be specifically located in the carboxysome to catalyze the generation of C02 for carboxylation (see ref. 20). However, apart from the observation that carboxysome-containing fractions purified from sucrose density gradients possess detectable CA activity (4), there has been no direct, unequivocal evidence for the specific location of CA activity with carboxysomes. This article deals with a reappraisal of the properties of intracellular carbonic anhydrase in the cyanobacterium Synechococcus PCC7942. This was prompted by the discovery that reduced DTT, a common antioxidant used in enzyme extraction buffers, causes inactivation of CACb,. Much of the previous work on intracellular CA (4) had, therefore, seriously underestimated its properties and specific activity. In this article, we have developed a new method for isolating carboxysomes and we show that a specific and major proportion of the cell's intracellular CA is located with the carboxysome fraction. We also report on the properties of this CA IL

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Mg + Concentration (mM) Figure 3. The stimulatory effect of increasing Mg2+ concentrations on the CA activity in carboxysome preparations (as percent of maximum rate). The complete curve was obtained with MgSO4 (-). Single data points are plotted for 20 mM MgCI2 (M) and 20 mm MgSO4 plus 40 mm NaCI (O). Prior to injection into the cuvette, the TP pellet preparation had been resuspended in Tes-EDTA buffer containing no Mg2+. Maximum activity was 120 units -mL1.

MgCl2 (results not shown). The nature of the divalent interaction is not known, but since Mg2' and Ca2+ share some chemical similarity and have similar stimulatory effects, the interaction may be moderately specific. More extensive studies with divalent cations have yet to be performed. Clearly, of the salts tested, MgSO4 gave the highest stimulation of CA activity, and accordingly, subsequent CA assays were done in the presence of 20 mM MgSO4. In the absence of Mg2+, carboxysome preparations had around 25% of maximum activity (Fig. 3). A significant proportion of this activity appears to correlate with the residual activity that was unaffected by DTT. Also of note is the observation that during CA assays in the presence of 20 mM MgSO4, activity in carboxysome preparations took up to 5 min to reach a steady maximum rate. This slow activation appears to be a property of the enzyme and may have been due to the fact that for these experiments, carboxysomes were initially suspended in a TesEDTA buffer containing no Mg2". The CA activity associated with carboxysomes was found to be very sensitive to the CA inhibitor EZ, with 80% of total activity being inhibited by the addition of 30 jAM EZ (Fig. 4). A residual activity of around 15% remained even in the presence of 600 ,M EZ. Since the residual activity was insensitive to EZ and DTT and did not require Mg2", it seems likely that this activity may be nonenzymic in nature. Even when carboxysome preparations were heated at 950C for 5 min, a residual activity was still evident (results not shown). If the residual activity is subtracted from the data in Figure 4, it is possible to calculate that the concentration of EZ necessary to inhibit 50% of CAcbx activity (I5o) is approximately 4 jiM. As a diagnostic test for the proportion of EZ-sensitive CA activity in crude extracts, an EZ concentration of 30 FM was

used (see below). From here on, we refer to this EZ-sensitive CA activity as 'low I50 CA activity. The effect of Ci concentration (as "8O-labeled "3Ci) on the measured level of CA activity in carboxysome preparations is depicted in Figure 5. These data indicate that CA activity is close to saturation at 1 mm Ci. Thus, measurements made at this level of Ci should be a good estimate of CACbX activity in vivo, where the cytosolic Ci concentration may exceed 20 mm. Subsequent measurements were made at 1 mm Ci. Extracts from cells grown at high Ci (2% C02) and low Ci (air-sparged Roux bottles) displayed two types of CA activity (Fig. 6). Around 60 to 70% of the total CA activity was inhibited by 30 ltM EZ. This low '5o CA activity would appear to be that which is associated with purified carboxysomes (Fig. 4). A second component of the CA activity in crude extracts was inhibited by 200 to 300 ZlM EZ, with an approximate I50 of 150 to 160 AM (Fig. 6). This 'high I5o' CA activity remains active in the presence of DTT, and would appear to be the same as the intracellular CA activity that has been previously characterized in crude extracts and purified fractions from Synechococcus PCC7942 (4). Previously, this CA activity was characterized as having an I50, for EZ, of 50 to 100 AM (4), but this now appears to be an underestimate due to the presence of some low I50 CA activity that had escaped DTT inactivation. The relative proportion of high 150 CA activity in crude extracts from low Ci cells was slightly higher than in high C1 cell extracts (Fig. 6). This appears to be related to the inducible nature of this component when cells are grown at limiting Ci levels (see below). Crude extracts also displayed a residual component of total CA activity (around 15%) that was unaffected by up to 600 AM EZ. Badger and Price (4) also reported the presence of a residual and apparently nonenzymic CA activity in extracts from Synechococcus PCC7942.

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PRICE ET AL.

of this second CA activity are similar to that of the C, pump in intact cells of Synechococcus PCC7942 (16, 17), and also the CA activity in crude extracts and pelletable fractions that have been previously characterized (4). Since the C1 pump is postulated to incorporate a CA-like step (17), we are currently

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Plant Physiol. Vol. 100, 1992

12 14

investigating the possibility that high I50 CA activity may be associated with the Ci transport system. From Table I it is also apparent that the ratio of low I50 CA activity to Rubisco activity changes with the C1 conditions during growth. On the assumption that carboxysome dimensions are the same under different C1 conditions, this would indicate that the coordinate control of CAcb. and Rubisco expression is not fixed, because carboxysomes from low C1 cells commonly had a CA to Rubisco ratio over 3 times greater than carboxysomes from high C1 cells (Table I). Also of note is the fact that the relative proportion of high 150 CA (as a percent of combined CA activity) increased around 2fold with growth under low Ci conditions.

13C. Concentration (mM) Effect of Culture Conditions on CA,b. Activity Figure 5. The effect of C, concentration (as "8O-labeled "3Ci) on the CA activity in carboxysome preparations (as percent of maximum rate). A 25-,uL aliquot of TP pellet containing a maximum CA activity of 176 units * mL-1 was added to the cuvette for each assay.

Carboxysome Recovery Experiments Recovery experiments were undertaken to determine if the majority of the cell's Rubisco and low I5o CA activities were associated with purified carboxysomes (i.e. the TP pellet). Table I shows the results of recovery experiments performed with cell extracts produced from cultures grown under several Ci regimes. CA and Rubisco activities were measured in both crude extracts and centrifuged extracts, as well as in TP pellets. However, since crude extracts contain some unbroken cells, the spun extract was taken as the starting point for determining the percent recovery in the purified TP pellet (Table I). It should be noted, however, that there was often up to a 15% loss of CA (low I5o) and Rubisco activities between the crude extract and spun extract stages. Notwithstanding, it can be seen that the TP pellet for both cell types contained most (75-90%) of the Rubisco and low o50 CA activities that were present in the spun extract (Table I). The TP pellet was also low in high I5o CA activity, especially when one takes in account the fact that nearly half of the high Io5 CA activity was a residual and possibly nonenzymic CA activity (Fig. 6). It was not routinely possible to assess the exact magnitude of the high I50 CA activity versus residual activity because of the difficulty in removing EZ carryover when 600 AM EZ was added to the cuvette. The most important result from the recovery experiments is the clear parallel relationship in the magnitude of the recovery of low I50 CA activity and Rubisco activity. Since the TP pellet is enriched in Rubisco-containing carboxysomes, it would be reasonable to propose that the low I50 CA activity is closely associated with the carboxysome. The low proportion of high I50 CA activity in the TP pellet suggests that this second component of the cell's CA activity is not normally associated with carboxysomes. The EZ inhibition properties

The relative activities of low 6 and high I50 CA activities and Rubisco activity were compared for cells grown at different Ci conditions. The results are shown in Table II. High Ci cells typically had low I5o and high I50 CA activities of around 30 to 34 and 10 to 12 units-mg-' Chl, respectively. Both these activities tended to fall in denser, late cultures grown at high Ci (Table II). The low I50 CA activity to Rubisco ratio for high Ci cells was typically 8 to 9. There was a clear induction of low I5o and high I50 CA activities after growth of cells at low Ci (air-sparged Roux bottle), with both components increasing around 3-fold. Unfortunately, overnight

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Ethoxyzolamide (gM) Figure 6. CA activity in crude extracts, as percent of maximum rate, plotted against the concentration of the CA inhibitor EZ in the assay. Crude extracts had maximum CA activities of 126 units- mL(100 ,L injected) and 46 units-mLV1 (200,L injected) for low C, cells and high C, cells, respectively.

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CARBOXYSOMAL CARBONIC ANHYDRASE ACTIVITY

Table I. Recovery Experiments for CA and Rubisco Activities Cell Type

Total High 150 (EZ)a CA Activity

Total Low 150 (EZ)b CA Activity

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Low 150 CA Activity as a Percent of Combined Activity

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High C, cells (2% CO2 grown) 14.2 81.9 107.8 119.1 21.6 Crude extract 12.2 100 80.0 101.3 20.3 Spun crude 11.2 96.2 85.5 97.4 14.1 TP pellet Low Ci cells (Air grown) (Roux) 8.2 121 214.7 60.4 81.1 Crude extract 6.9 100 62.0 67.4 177.5 Spun crude 5.2 78.1 91.3 138.6 12.1 TP pellet a CA activity remaining in the presence of 30 ,M EZ (mostly composed of CA activity with 150 = 150-160 MM EZ). by the addition of 30 MM EZ (i.e. CA activity with 150 = 4 IAM EZ).

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118 26.2 100 25.6 26.5 75.4 b CA activity abolished

catalytic conversion by a factor of 32,000 and 149,000 times, respectively (Table II). It is also apparent from the data in Table II that high 150 CA activity is induced by growth of cells under low C, conditions. This would be consistent with the possibility that this CA activity may be associated with the C1 pump, which is presumably located on the plasma membrane. Attempts to detect CA activity in purified plasma membrane preparations have so far been negative (4; G.D. Price and C.A. Howitt, unpublished results). Such preparations are usually produced on sucrose floatation gradients in the presence of EDTA, with preparation time being up to 18 h (15). Such treatment may be inappropriate for maintaining any CA activity associated with a membrane-bound Ci pump, and more suitable protocols may need to be developed. With the discovery of a CA activity that is clearly associated with carboxysomes, it was of interest to reevaluate one of our high C02-requiring mutants, Type II mutant No. 68 (19), for CA