Plant Carbonic Anhydrases

Abstract

Carbonic anhydrase (EC.4.2.1.1) was purified from leaves of the dicotyledon Pisum sativumrl L. (56-fold) and from leaves of the monocotyledon Tradescantia albiflora . The molecular weight of the Pisumn enzyme was estimated to be 188,000 + 8,000 with subunit sizes of 28,000 ± 3,000 and 56,600 + 3,500. It contained 1 mole zinc per 32,500 + 2,000 g protein. MATERIALS AND METHODSPlant Material. Leaves were taken from Tradescantia albiflora Kunth. which was growing in the field during the southern hemisphere summer. The leaves and stem were taken from 2-to 3-week-old plants of Pisumn sativumi L. cv. Greenfeast (Yates Seeds, Sydney, N.S.W.) which were grown in vermicuLlite in a glasshouse.Buffer Solutions. Unless stated otherwise, the pH value of all buffers was determined at 5 C and all contained 1 mM Na,-EDTA and 0.1 M 2-mercaptoethanol. For enzyme isolation, two buffers at pH 8.3 were used; Buffer A contained 0.3 M tris-SO and buffer B contained 10 mm tris-SO.Isolation of Carbonic Anhydrase from Tradescantia. All operations were carried out at 1 C. Nine kg of leaves were chilled and thoroughly homogenized in a Waring Blendor with 9 liters of buffer A. The homogenate was filtered through two layers of nylon mesh (60 tim hole size; Nycloth Co., Harris Park, N.S.W.) and centrifuged at 35,000g for 40 min. The pellet was discarded, and finely powdered ammonium sulfate was added to the supernatant (0.28 g to each ml) with continuous stirring. After centrifugation (35,000g for 30 min) the pellet was discarded. More ammonium sulfate was added to the supernatant (0.16 g to each ml) and the precipitated enzyme recovered by centrifugation (35,000g for 30 min). The pellet was dissolved in the minimum volume of buffer B and dialyzed against three changes (each 4 liters) of 5 mm tris-SO, buffer, pH 8.3, containing 20 mm ME.4 The ME concentration of the dialysate was adjusted to about 0.1 M, and insoluble material was removed by centrifugation (20,000g for 15 min). The supernatant (245 ml) was mixed with A-50 DEAE-Sephadex (20 g dry weight equilibrated with buffer B) in a Buchner funnel and eluted with 1.2 liters buffer B containing 10 mm Na,SO,. This effluent was discarded, and the enzyme was eluted with 1.2 liters buffer B containing 100 mm Na.,SO,. Following ammonium sulfate precipitation (0.44 g added to each ml) the preparation was dissolved and dialyzed as above. The dialysate was placed on a 2-X 30-cm column of DEAEcellulose (Whatman DE-32) equilibrated with buffer B containing 10 mm Na,SO,, and the enzyme was eluted (see Fig. IA) at 25 ml per hr with a linear Na,SO, gradient (250 ml buffer B with 10 mm Na,SO, in the mixing chamber and 250 ml buffer B with 200 mm Na,SO, in the reservoir). The "active' fractions (within the arrows shown in Fig. 1 A) were pooled, concentrated by ammonium sulfate precipitation (0.44 g added to each ml) to 20 ml, and placed on a 36-x 2.5-cm 'Abbreviations: DEAE: diethylaminoethyl; ME: 2-mercaptoethanol; SDS: sodium dodecylsulfate.

Carbonic anhydrases in plants and algae

“…Monocot CAs have not been characterized using physical methods. A CA from Tradescantia leaves, presumably the chloroplast b-CA, appears to be a monomer or dimer when subjected to gel filtration (Atkins, Patterson & Graham 1972). The chloroplast b-CA from barley appears to be a dimer or trimer using the same method (M.H.…”

The Mechanistic Basis of Internal Conductance: A Theoretical Analysis of Mesophyll Cell Photosynthesis and CO2 Diffusion

“…Atkins et al (1972a) determined the total CA activity in extracts from a range of species. Using a specific activity of 23,900 units mg 21 (Atkins et al, 1972b), a molecular mass of 28 kD per active site, and a chloroplast volume of 30 mm mg 21 chlorophyll, it can be calculated that the CA concentration in these species was between 0.04 and 0.69 mol active sites m 23 . Alternatively, the CA concentration can be estimated from measurements of in vivo CA activity (Price et al, 1994;Peltier et al, 1995;Gillon and Yakir, 2000).…”

“…For the purpose of this model, we assumed a C 3 leaf with chloroplasts containing 1.25 mM Rubisco sites, an electron transport capacity of 90 mmol m 22 s 21 , a chloroplast CO 2 compensation point of 4.1 Pa, and a dark respiration rate of 0.8 mmol m 22 s 21 (Table I). The concentration of CA was based on in vitro and in vivo activity measurements (Atkins et al, 1972a(Atkins et al, , 1972bPrice et al, 1994;Peltier et al, 1995;Gillon and Yakir, 2000). Atkins et al (1972a) determined the total CA activity in extracts from a range of species.…”

“…Traditionally, the r i has been divided into two major components : the intercellular air space resistance (from substomatal cavities to the cell surface) and the liquid-phase resistance (from the cell walls to the site of carboxylation). Theoretical and Atkins et al (1972aAtkins et al ( , 1972b; Price et al (1994); Peltier et al (1995); Gillon and Yakir (2000) experimental results suggested that the resistance of the intercellular air spaces is negligible in many cases (Genty et al, 1998;Aalto and Juurola, 2002;Piel et al, 2002), although it accounts for a significant portion of r i in thick, hypostomatous leaves (Parkhurst and Mott, 1990;Parkhurst, 1994). The resistances of the cell wall (25%-50%) and chloroplast envelopes (24%-76%) are considered to make major contributions to the total r i Gillon and Yakir, 2000).…”

Plant Carbonic Anhydrases

“…In this respect also exlr_2cs from T. albiflora and P. sativurn which were freshly prepared did not differ from extracts stored at 5 C for up to 2 weeks. P.rtially purified preparations from these two species were identical with the crude extracts (2).…”