Carbonic anhydrase and CO2 fixation in isolated chloroplasts

(5,11,12,15). These activity differences between species may be as great as 18-fold, although in some cases there may only be a 2-or 3-fold greater activity. One exception has been described in extracts of Amaranthus edulis, which had carbonic anhydrase activity similar to that of spinach in one experiment (11). However, A. viridis and A. palmeri had considerably lower activities.In spinach leaves most of the carbonic anhydrase is found in the chloroplast (12,20), where it is located in the stromal fraction (20). But in maize, it was found to be a cytoplasmic enzyme (12), although the activity in the preparation was low.The complete extraction of enzymes from leaves such as maize is not always easily accomplished (1,3,24) Enzyme Extraction. Leaves from young plants (1-2 weeks) were picked and used immediately for extraction. Whole leaves (2 g) were placed in a chilled mortar (4 C), and 1 g of sharp sand was added. When expanding leaves of plants 6 to 8 weeks old were used, the main ribs were removed and the blades were cut into 2-cm strips crosswise prior to grinding. The following progressive grinding procedure was based on the method of Bucke and Long (3).Twenty-five ml of a medium (4 C) containing 50 mM veronal buffer, pH 8.2, 1 mm EDTA, and 10 mM 2-mercaptoethanol were added and the contents were gently ground under a stream of nitrogen gas until the solution was light green. This was filtered through six layers of cheesecloth, the residue was washed with 5.0 ml of medium, and the filtrates were combined. The residue was returned to the mortar and 10.0 ml of medium was added. Grinding under nitrogen was continued until the tissue appeared translucent. The contents were filtered through the same cheesecloth, washed, and the residue returned to the mortar. The grinding, filtering, and washing were repeated twice more for a total of four grindings. Filtrates were usually combined into one vessel under nitrogen, unless separate extracts for each grinding were collected.Enzyme extracts were assayed within 30 min of preparation. A stream of nitrogen was blown over the extracts throughout the operation.Fractionation of Leaves. As described by Bucke and Long (3) and based also on microscopic examination of the chloroplast types in each successive extract, the combined filtrates from the first and second grinding were taken together when an enzymic extract of mesophyll cells was desired. The combined solutions from the third and fourth grindings were used if an enzymic extract of the bundle-sheath cells was needed.Five fields each containing 100 to 300 chloroplasts were counted for each fraction in three separate experiments. Preparations consisting mainly of mesophyll or bundlesheath cells were also obtained by the differential grinding method of Woo et al. (23). The isolation medium consisted 336 www.plantphysiol.org on May 9, 2018 -Published by Downloaded from

Section: Resultsmentioning

confidence: 99%

The Distribution of Carbonic Anhydrase and Ribulose Diphosphate Carboxylase in Maize Leaves

Poincelot

1972

“…This has been suggested by many investigators (7,11,21,29,30). Two things must be demonstrated before this suggestion can be considered valid: (a) the uncatalyzed rate of CO2 formation from the HCO2-pool must be less than the rate of photosynthesis and (b) the amount of bicarbonate in the pool must be known.…”

Section: Resultsmentioning

confidence: 94%

“…The results on inhibition by acetazolamide (30) (5, 6, 8, 10, 18). Another is that it aids in concentrating CO2 in the chloroplast or that it causes the rapid conversion of HCO3-to the active species of carbon used by ribulose diphosphate carboxylase (7,11,21,24,31). In the discussion to follow, we will be able to rule out the participation of carbonic anhydrase in those reactions requiring the diffusion of CO2 or its production from bicarbonate and support the contention that carbonic anhydrase could aid photosynthesis by transiently preventing drastic changes in pH or by binding or hydrating compounds other than CO2.-Diffusion of CO2.…”

Section: Resultsmentioning

confidence: 99%

“…The observation that an increased CO2 concentration increased photosynthesis fostered, in part, the suggestion that carbonic anhydrase enhanced fixation by increasing the intrachloroplastic levels of CO2 (7,11,21,24,31). More recently it has been shown that the Kini of CO2 for ribulose diphosphate carboxylase was similar to the concentration of CO2 that was in a solution which was in equilibrium with air (3).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Carbonic Anhydrase of Spinach

Jacobson¹,

Fong²,

Heath³

1975

Carbonic anhydrase activity was determined in spinach (Spinacia oleracea) leaf organelles isolated on sucrose density gradients and was found to be predominantly in the intact chloroplast fraction. The small amount of activity associated with the mitochondrial fractions was probably due to intact chloroplast contamination. No activity could be associated with the broken chloroplast or microbody fractions. Based upon inhibitor studies, carbonic anhydrase was found to be around 2 mM in the chloroplast. Ethoxzolamide, an inhibitor of carbonic anhydrase, reduced C02 fixation in intact chloroplasts. The concentration required to inhibit CO2 fixation 20 to 40% was in excess of that required to inhibit the purified enzyme. The inhibition was partially reversed by C02. Ethoxzolamide had no effect on photosynthetic NADP reduction or photophosphorylation measured by methyl viologen reduction. The In addition, the finding that acetazolamide inhibited photosynthetic electron transport as well as carbonic anhydrase seriously questions whether or not the acetazolamide inhibition of photosynthetic CO2 fixation was a function of carbon anhydrase activity. This paper is further concerned with the study of another inhibitor or carbonic anhydrase-ethoxzolamide-with respect to its inhibitory kinetics and its effect upon isolated intact chloroplasts. MATERIALS AND METHODSThe leaves of washed, commercial spinach (Spinacia oleracea), after deribbing, were chopped gently with razor blades in a grinding medium of 400 mm sorbitol, 20 mm HEPES buffer (pH 8.0), 10 mm MgNa2-EDTA, 1 mM dithiothreitol, and 1% bovine serum albumin at 0 C (1.5 ml of grinding medium per g fresh weight). The leaf homogenate was then gently ground in a mortar and pestle at 0 C which contained about 0.5 ml of partially frozen grinding medium per g fresh weight of leaves.The slurry was then filtered once through a 30-Mtm diameter nylon mesh to remove cell wall debris and unbroken cells. The supernatant of this solution obtained from a low speed centrifugation (either 1,000g or 3,000g) was then layered onto sucrose density gradients, as previously described (24), with the following modifications. Pellets were resuspended in grinding medium without MgNa2-EDTA, and 1 mM dithiothreitol was substituted for mercaptoethanol. The gradients were centrifuged at 25,000 rpm in a Beckman SW27 swinging bucket rotor in a Beckman L2-50 ultracentrifuge.Isolated intact spinach chloroplasts, used for the inhibitor studies, were prepared as previously described (12). Published methods were used to assay for glyceraldehyde-3-P dehydrogenase, malate dehydrogenase (24), and chlorophyll concentrations (1). The carbonic anhydrase assay consisted of a reaction mixture of 4.0 ml of barbituric acid buffer (22.5 mM at pH 9.0), 0.1 ml of enzyme suspension, and 0.9 ml of distilled water. Blanks were either 0.1 ml of boiled enzyme suspension or distilled water. The reaction was followed by recording the pH changes on a Radiometer pH meter (type TTTlc) and a Bausch and Lomb recorder. The ...

“…All the mammalian carbonic anhydrases (EC 4 (8). Partially purified (5,6,18) and highly purified (12, [14][15][16] preparations of carbonic anhydrase have been made from the leaves of a number of plants.…”

mentioning

confidence: 99%

Plant Carbonic Anhydrases

Atkins¹,

Patterson

Graham

1972

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.