plant carbonic anh-vdrases · 2020. 1. 18. · stability in solution, and sensitivity to...

Plant Physiol. (1972) 50, 218-223

Plant Carbonic Anh-vdrasesII. PREPARATION AND SOME PROPERTIES OF MONOCOTYLEDON AND DICOTYLEDON

ENZYME TYPES

Received for publication December 1, 1971

C. A. ATKINS,1' B. D. PATTERSON,' AND D. GRAHAMPlant Physiology Unit, Commonwealth Scientific and Industrial Research Organization, Division of FoodResearch, and School of Biological Sciences, Macquarie University, Northt Ryde, 2113, Sydney, 4ustralia

ABSTRACT

Carbonic anhydrase (EC.4.2.1.1) was purified from leavesof the dicotyledon Pisum sativumrl L. (56-fold) and from leavesof the monocotyledon Tradescantia albiflora Kunth. (24-fold).The molecular weight of the Pisumn enzyme was estimated to be188,000 + 8,000 with subunit sizes of 28,000 ± 3,000 and56,600 + 3,500. It contained 1 mole zinc per 32,500 + 2,000 gprotein. The molecular weight of the Tradescantia enzyme wasestimated to be 42,000 + 2,000 with a subunit size of 27,500 +

2,200. It contained 1 mole zinc per 34,000 + 2,000 g protein.The two enzyme preparations were different in specific activity,stability in solution, and sensitivity to sulfonamides and inor-ganic anions. Gel electrophoresis separated each purified prep-aration into two active enzyme bands.

All the mammalian carbonic anhydrases (EC 4.2.1 . 1)which have been studied are single polypeptide chains with amolecular weight around 30,000 and contain 1 atom of zincper protein molecule (8). Partially purified (5, 6, 18) and highlypurified (12, 14-16) preparations of carbonic anhydrase havebeen made from the leaves of a number of plants. These en-zymes are larger than those from animal tissues, the reportedmolecular weight being 180,000 for carbonic anhydrase fromPetroselenium crispum (parsley) (16) and 140,000 fromSpinacia oleracea (spinach) (12). Zinc was found in some(14-16, 18) but not all (5, 12) preparations.

In an electrophoretic study of leaf extracts from manyplant species, we have shown (2) that while the carbonicanhydrases from dicotyledons are similar in size to the iso-lated parsley (16) and spinach (12) enzymes, those frommonocotyledons are closer in size to the mammalian carbonicanhydrases.

In the present study carbonic anhydrases were isolated fromthe dicotyledon Pisuin sativumt L. and the monocotyledonTradescantia albiflora Kunth. The molecular weights, subunitsizes, zinc content, sensitivity to inhibitors, and stability ofthese preparations were determined and compared.

'Recipient of Rothman's post doctoral fellowship.2 Present address: % International Atomic Energy Agency, P.O.

Box 645, A-1011 Vienna, Austria.'Present address: East Malling Research Station, East Malling,

Maidstone, Kent, U.K.

MATERIALS AND METHODS

Plant Material. Leaves were taken from Tradescantia albi-flora Kunth. which was growing in the field during the south-ern hemisphere summer. The leaves and stem were taken from2- to 3-week-old plants of Pisumn sativumi L. cv. Greenfeast(Yates Seeds, Sydney, N.S.W.) which were grown in vermicuL-lite in a glasshouse.

Buffer Solutions. Unless stated otherwise, the pH value ofall 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 Mtris-SO and buffer B contained 10 mm tris-SO.

Isolation of Carbonic Anhydrase from Tradescantia. Alloperations were carried out at 1 C. Nine kg of leaves werechilled and thoroughly homogenized in a Waring Blendor with9 liters of buffer A. The homogenate was filtered through twolayers of nylon mesh (60 tim hole size; Nycloth Co., HarrisPark, N.S.W.) and centrifuged at 35,000g for 40 min. Thepellet was discarded, and finely powdered ammonium sulfatewas added to the supernatant (0.28 g to each ml) with con-tinuous stirring. After centrifugation (35,000g for 30 min) thepellet was discarded. More ammonium sulfate was added tothe supernatant (0.16 g to each ml) and the precipitated en-zyme recovered by centrifugation (35,000g for 30 min). Thepellet was dissolved in the minimum volume of buffer B anddialyzed against three changes (each 4 liters) of 5 mm tris-SO,buffer, pH 8.3, containing 20 mm ME.4 The ME concentrationof the dialysate was adjusted to about 0.1 M, and insolublematerial was removed by centrifugation (20,000g for 15 min).The supernatant (245 ml) was mixed with A-50 DEAE-Sepha-dex (20 g dry weight equilibrated with buffer B) in a Buchnerfunnel and eluted with 1.2 liters buffer B containing 10 mmNa,SO,. This effluent was discarded, and the enzyme waseluted with 1.2 liters buffer B containing 100 mm Na.,SO,. Fol-lowing ammonium sulfate precipitation (0.44 g added to eachml) the preparation was dissolved and dialyzed as above. Thedialysate was placed on a 2- X 30-cm column of DEAE-cellulose (Whatman DE-32) equilibrated with buffer B con-taining 10 mm Na,SO,, and the enzyme was eluted (see Fig.IA) at 25 ml per hr with a linear Na,SO, gradient (250 mlbuffer B with 10 mm Na,SO, in the mixing chamber and 250ml buffer B with 200 mm Na,SO, in the reservoir). The "active'fractions (within the arrows shown in Fig. 1A) were pooled,concentrated by ammonium sulfate precipitation (0.44 gadded to each ml) to 20 ml, and placed on a 36- x 2.5-cm

'Abbreviations: DEAE: diethylaminoethyl; ME: 2-mercaptoeth-anol; SDS: sodium dodecylsulfate.

218

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PLANT CARBONIC ANHYDRASES. II

column of G-100 Sephadex. The enzyme was eluted with buf-fer B at 25 ml per hr, and the active fractions were pooled(within the arrows shown in Fig. 2A) and stored as an am-monium sulfate precipitate in the presence of excess sulfhydrylreagent (0.1 M ME or 15 mm dithiothreitol). The suspensionwas adjusted to pH 8.3 by adding solid tris.

Isolation of Carbonic Anhydrase from Pisum. Leaf andstem tissue (3.3 kg) was homogenized, and the soluble extractwas recovered as in the preparation for Tradescantia. Theammonium sulfate fractionation was carried out as above ex-cept that first 0.2 g was added to each milliliter of the extractsupernatant and after centrifugation a further 0.2 g was addedto each milliliter to precipitate the enzyme. Following dialysis,the preparation was mixed with DEAE-Sephadex (see above)and eluted with 1 liter buffer B. Unlike the Tradescentia en-zyme, which was firmly bound to the DEAE-Sephadex, mostof the Pisumn carbonic anhydrase activity was recovered withthis eluate. The eluate was diluted to 2 liters with buffer A andfinely powdered ammonium sulfate added (0.26 g to each ml)with continuous stirring. After 24 hr the precipitate was re-moved, and more ammonium sulfate (0.7 g to each ml) wasadded to the supernatant to recover the enzyme (see above).The preparation was dialyzed and chromatographed onDEAE-cellulose. The same conditions were used as with theTradescantia enzyme except that the Na2SO, gradient was lesssteep (see Fig. 1B); the mixing vessel containing 3 mm Na2SO4

75

(21-

0

0,25

80

2

80

2 60

z

4cm 40

20

1 5

1*0

E

_

05

- 3'

Eo

2

8EEo0u0

4w

1-

0

0-4

0 2

.E

2 -Cwi

szO1 ,w

gomx-K

EFFLUENT (ml)

200 400

EFFLUENT (ml)

FIG. 1. Elution of (A) Tradescantia and (B) Pisum carbonic an-hydrase activity from a DEAE-cellulose column (2 X 30 cm) witha linear gradient of Na2SO4 in pH 8.3, 10 mM tris buffer. Thosefractions within the arrows at the top were combined in the purifica-tion procedure.

E

..4

(A

>. 10zc

z0

-E

0-

z0u

02

I

100

50

01100 fI ICY

50

100 200

EFFLUENT (ml}

FIG. 2. A: Elution of Tradescantia carbonic anhydrase activityfrom a G-100 Sephadex column (2.5 X 36 cm); B: elution of Pisumcarbonic anhydrase activity from a G-200 Sephadex column (2.5 X36 cm) with pH 8.3 10 mm Tris-SO4 buffer. Those fractions withinthe arrows at the top were combined in the purification procedure.

and the reservoir 100 mm Na2SO, in 300 ml buffer B. The"active" fractions (within the arrows shown in Fig. 1B) werepooled and concentrated to 3 ml by ammonium sulfate pre-cipitation (0.4 g added to each ml). The redissolved enzymewas applied to a 36- x 2.5-cm column of G-200 Sephadex andeluted at 25 ml per hr with buffer B. The active fractions were

pooled (within the arrows shown in Fig. 2B) and stored in thesame way as the Tradescantia preparation.

Determination of Molecular Weight by Gel Filtration. Theelution volume (Fig. 3) of purified Tradescantia carbonicanhydrase was compared with those of ovalbumin (gift fromMr. M. B. Smith, C.S.I.R.O. Division of Food Research, NorthRyde, N.S.W.), bovine /3-lactoglobulin (Nutritional Biochemi-cals Corporation), and bovine erythrocyte carbonic anhydrase(Sigma Chemical Co.). The proteins were eluted at 25 ml perhr with 50 mm tris-SO buffer, pH 8.3, containing 200 mM

Na2SO, from a 36- x 2.5-cm column of G-100 Sephadex.The elution volume for Pisum carbonic anhydrase was

compared with those of bovine y-globulin (Sigma), yeast alco-hol dehydrogenase (Sigma), and yeast hexokinase (Type III,Sigma) from a 36- x 2.5-cm column of Agarose (Bio-GelA-1.5m). Elution was at 25 ml per hr with the buffer used forthe Tradescantia preparation.

Sodium Dodecyl Sulfate Gel Electrophoresis. Five-milli-gram samples of bovine serum albumin (crystalline, Sigma),pepsin (Sigma), ovalbumin, /8-lactoglobulin, Pisum carbonicanhydrase, and Tradescantia carbonic anhydrase were dena-

200100

EFFLUENT ( ml)

Plant Physiol. Vol. 50, 1972 219



ATKINS, PATTERSON, AND GRAHAM

5

40

x

3: 3-j

0

2

130 140

20

10150 200

EFFLUENT VOLUME (ml)

FIG. 3. Determination of the molecular weight of plant carbonic anhydrases (CA) by gel filtration. The Tradescantia enzyme was eluted from

a 36- X 2.5-cm column of G-100 Sephadex, and the Pisun enzyme from a 36- X 2.5-cm column of Bio-Gel A-1.5m with pH 8.3, 50 mm trisbuffer.

tured and reduced by the method of Shapiro et al. (13). Thepreparations were subjected to electrophoresis for 5 hr at 50 v

with 25 mm, pH 7.2, phosphate buffer containing 0.1% w/vSDS and 0.1% v/v ME using 7 x 7 cm, 10% polyacrylamideslabs in the apparatus of Margolis and Kenrick (9). Proteinbands were detected by staining with 0.38% w/v amido blackin 1 M acetic acid.

Polyacrylamide Gradient Gel Electrophoresis. Samples ofcrude extract as well as purified carbonic anhydrases were

subjected to electrophoresis and the enzyme activity detectedon gels as described previously (10).

Determination of Sedimentation Velocity. The sedimenta-tion velocity of purified carbonic anhydrase from Tradescantia(4 or 8 mg protein per ml in 1 mm tris-S04 buffer, pH 8.3,containing 50 mm Na2SO,4 and 5 mm ME) was determined at59,780 rpm and 20 C in a Spinco Model E ultracentrifuge.

Determination of Zinc. Usually 4 ml of solution containingabout 1 mg of protein was taken, and 0.5 ml of 60% v/vperchloric acid added. The mixture was evaporated almost todryness on a sand bath and then dissolved to a final volume of4 ml with 0.1 N HCI. Standard samples containing from 0.1 to1 ,ug of Zn/ml were carried through the same digestion pro-cedure. Zinc was measured by atomic absorption spectroscopy.In the calibration range used (up to 1 ,tg Zn per ml) the re-

sponse was linear (1 absorbance unit = 1.43 ,tg Zn/ml).Determination of Protein. For specific activity and specific

zinc content estimations the method of Lowry (7) was usedwith bovine serum albumin (Sigma, crystalline) as a standard.

Table I. Purificationt of Tradescanitia Carboniic Anzhydrase

Enzyme Fraction Protein Enzyme Specific Recov-Activity Activity cry

mzg/mil ,units/mitl units/mg %

Homogenate supernatant 1.05 1,870 1,780 100(NH4)2SO4 fractionation and 24.30 85,500 3,520 76

dialysisPooled "active" fractions from 5.00 51,150 10,200 40DEAE-Sephadex and dia-lysis

Pooled "active" fractions from 0.142 5, 530 38,900 36DEAE-cellulose column

Pooled "active" fractions from 0.146 6,180 42,300 30

G-100 Sephadex column

The absorbance at 280 nm was used to monitor protein in thecolumn effluents with standard proteins or partially purifiedcarbonic anhydrase preparations.

Carbonic Anhydrase Assay. The enzyme activity (units)was assayed by the colorimetric method of Wilbur and Ander-son (20) as used by Rickli et al. (1 1).

Inhibitor Studies. Sodium acetazolamide (Lederle Labora-tories, Derrimut, Victoria) and ethoxyzolamide (Upjohn Pty.Ltd. Rydalmere, N.S.W.) were used in solution with 95% v/vethanol. Potassium nitrate and sodium azide were used in

aqueous solution. From 1 to 10 ,ul of inhibitor solution was

added to the veronal-indicator buffer used in the usual enzyme

assay (11) and held at 0.5 C. Then a 10-1-l sample of enzyme

was added, and after 2 min at 0.5 C the reaction was started bythe addition of CO2-saturated water. For each compound theconcentration causing 50% inhibition was found from a plotof the log concentration and the observed percentage of inhibi-tion. Carbonic anhydrase activity was unaffected by the etha-nol added with the sulfonamides.

Stability. Samples of each enzyme were held at 5 C with 0.1M ME at different pH values in closed vessels. The buffers usedwere 0.1 M acetate at pH 4.85 and 5.4; 0.1 M tris-maleate at pH6.1, 7.2, and 8.25; 0.1 M tris-S04 at pH 8.3; and 0.1 M glycine-NaOH at pH 9.05, 10.25, and 11.3. The solutions were assayedperiodically for up to 10 days to determine the rate of loss ofcarbonic anhydrase activity.

RESULTS

Purification. The purification of the Tradescantia carbonicanhydrase is shown in Table I. There was a 24-fold purificationof the enzyme from the soluble leaf proteins, and 30% of theactivity assayed in the crude extract was recovered. The puri-fication of the Pisumn carbonic anhydrase is shown in TableII. There was a 56-fold purification of the enzyme from thesoluble leaf proteins, and 10% of the activity assayed in thecrude extract was recovered.

Molecular Weight Determinations. Figure 3 clearly showsthat the carbonic anhydrases from the two species were of dif-ferent molecular weight (by gel filtration). The Tradescantiaactivity eluted between ovalbumin (45,000) and /3-lactoglobulin(35,000) giving an estimate for molecular weight of 42,000 +2,000. The Pisum enzyme activity eluted between y-globulin(205,000, (1)) and yeast alcohol dehydrogenase (150,000) giv-ing an estimate of 188,000 8,000.

Ovalbumin

Tradescantia CA

beta - lactoglobulin

BovineerythrocyteCA

gamma globulin

Pea CA

Alcoholdehydrogenase

Hexoki

220 Plant Physiol. Vol. 50, 1972




The molecular weight of the protein in the Tradescantiapreparation was confirmed by a determination of sedimenta-tion velocity. A single, symmetrical peak was found in theultracentrifuge with a sedimentation constant around 3.2 S.

After denaturation both preparations contained a protein ofaround 27,000 mol wt in the presence of SDS (Fig. 4). ThePisum enzyme, however, yielded a second protein of abouttwice this size (52,000, Fig. 4). The values found from five gelswere 27,500 + 2,200 for Tradescantia and 28,000 + 3,000and 56,500 + 3,500 for Pisum carbonic anhydrase.

Zinc Content. The carbonic anhydrase from both specieswas associated with zinc in column effluents where elution ofthe metal was coincident with enzyme activity (Fig. 2). Duringthe preparation of these enzymes the ratio protein/Zn de-creased with increasing purity. The minimum protein/zincratio found in the purified enzymes was in each case about33,000 g protein per mole Zn (Fig. 2A and B). For foursamples of Tradescantia enzyme the minimum weight of pro-tein per mole Zn was 34,000 + 2,000 g protein, and for fivesamples of Pisum enzyme was 32,500 + 2,000 g.

Gel Electrophoresis. The difference in the size of thecarbonic anhydrases from the two species is correlated with theelectrophoretic mobility of enzyme activity in crude extracts (1and 2 in Fig. 5) on gradient gels. The two major bands ofactivity found in the homogenate from Tradescantia were alsopresent in the purified enzyme (2 and 3 in Fig. 5). A similarresult was obtained for the purified Pisumn carbonic anhydrase.

Table II. Putrificationi of Pisum Carboniic Anzhydrase

Enzyme Fraction Protein Enzyme Specific Recov-Activity Activity ery

mng/mIl uinits/ml units/mg %

Homogenate supernatant 8.15 3,600 431 100(NH4)2SO4 fractionation and 36.20 21,200 587 75

dialysisPooled "active" fractions from 4.52 6,000 1,330 56DEAE-Sephadex

(NH4)2SO4 fractionation and 22.15 112,500 5,080 26dialysis

Pooled "active" fractions from 1.88 32,550 17,320 18DEAE-cellulose column

Pooled "active" fractions from 1.40 33,400 23,900 10G-200 Sephadex column

10

o sx

3:-j

0

0 4 8

MIGRATION IN GEL (cm)

FIG. 4. Polyacrylamide gel electrophoresis of purified Trades-cantia and Pisum carbonic anhydrase (CA) after denaturation andreduction in the presence of 0.1% sodium dodecyl sulfate (see "Ma-terials and Methods").

221

00 0 c

0

=. @0>>

S. m^ l

CL a£L a 0 0

1 2 3 4 5 6 7 8FIG. 5. Photograph of low temperature ultraviolet fluorescence

due to carbonic anhydrase (CA) activity on developed polyacrylam-ide gradient gels (see Ref. 10). The samples were subjected toelectrophoresis from top to bottom. 1: Leaf homogenate fromPisum; 2: leaf homogenate from Tradescantia; 3: purified car-bonic anhydrase from Tradescantia; 4: same as 3 but after storageas an ammonium sulfate precipitate; 5 to 8: samples from aTradescantia preparation after ion exchange chromatography (seeFig. 1) being at 200, 220, 230, and 250 ml effluent, respectively.

Table III. Inzhibitionz of Purified Carbonzic An/hy-drases from Pisiuman2d Tradescantia Leaves by Sulfonzamides anid

Iznorgantic Anionis

Concn for 50% Inhibition ofICarbonicAnhydrase Activity

Inhibitor

Pisum Tradescantia

Acetazolamide 450 270Ethoxyzolamide 5 18Sodium azide 9 13Potassium nitrate 38 175

The trace of less mobile activity found in the homogenate ofTradescantia was removed during storage as an ammoniumsulfate precipitate.The form of the eluted activity found during ion-exchange

chromatography on the DEAE-cellulose column used (seeFig. 1, A and B) suggests that there was partial separation ofmore than one carbonic anhydrase in both preparations. Thiswas confirmed by gel electrophoresis of fractions from thecolumn effluent of the Tradescantia enzyme (5-8 in Fig. 5).When a strip of this gel-containing enzyme was removed andplaced at right angles on the origin of a fresh polyacrylamideslab and subjected to electrophoresis, each band yielded a

single band, suggesting that the two forms of the enzyme were

not interconvertible under the conditions used.Inhibition. The two enzyme preparations differed in their

sensitivity to the sulfonamide and inorganic anion inhibitorstested (Table III).

Stability in Solution. The Pisum carbonic anhydrase was

more stable in solution than was the enzyme from Tradescantia(Fig. 6). At pH 4.85 in each case the half-life of activity wasaround 1 min and at 5.4 around 10 min. At pH values greaterthan 6.0, however, the Pisum enzyme was up to 15 times morestable (pH 7.2 in Fig. 6) than that from Tradescantia. Both

Plant Physiol. Vol. 50, 1972

BSA

Pea CA

Ovalbum in

Pepsin

Pea CATradescantia CA

beta- lactoglobulin

1



ATKINS, PATTERSON, AND GRAHAM

>- ~~~AA

46z

4

0

A--~...A Pea

0

0

UA. 2

TradescantiaPetroselinum crispum cA

w

4 6 8 10 12

pH

FIG. 6. Stability of Pisum and Tradescantia carbonic anhydrases

(CA) in the presence of 0.1 m 2-mercaptoethanol and 0.1 m buffers

(see "Materials and Methods" for composition) at different pHvalues at 5 C.

Table IV. Comnparison of Some Properties of Puirified Carbontic

Aihydrases from Tradescantia, Pisuim, and

Petroselinum crispum Leaves

Source

Property - P

Tradescantia Pisum Petroselin(1

* ~ ~ ~~~~~~~~I iActivity (units g fresh wt of leaf)!, 3,300 7,600Purification X24 X56 X133Specific activity (units,/mg pro- 42,300 23,900 12,000

tein)Molecular weight 42,000 188,000 180,0002

±2,000' 48,000'Subunit molecular weight3 27,500 28,000 29,000

±2,200 ±3,000Specific Zn content (g protein," 34,000 32,500 29,000mole Zn) 2,000 ±2,000

'By gel filtration.2 By sedimentation equilibrium.I By SDS gel electrophoresis.

enzymes showed maximum stability near pH 8.25 and bothwere less stable with increasing alkalinity (Fig. 6).

DISCUSSION

The Pisumn enzyme with a molecular weight of about188,000 was similar to the parsley enzyme isolated by Tobin(16) (Table IV). The only other weight estimation of plantcarbonic anhydrase is 140,000 for a zinc-free enzyme isolatedfrom spinach leaves (12). As the electrophoretic migration ofcarbonic anhydrases from leaf extracts of species in 19dicotyledon families was similar to those from pea, spinach,and parsley on gradient gels (2), it may be inferred that the"dicotyledon-type" carbonic anhydrase has a molecular weightaround 180,000. The Tradescantia carbonic anhydrase wasmuch smaller than the dicotyledon-type enzyme. Whileestimates of weight are not available for any other species, the

electrophoretic similarity found for examples from 1 1 mono-cotyledon families (2) suggests a value around 40,000 for themonocotyledon-type enzyme.

Tobin (16) has proposed that the parsley enzyme is madeup of six similar subunits (mol wt 30,000), each containing 1atom of zinc. The two polypeptide chains found in SDS gelelectrophoresis of Pisum carbonic anhydrase might indicatetwo different sized subunits in this enzyme. However, aggrega-tion of protein monomers into dimers by disulfide bondingduring electrophoresis has been noted previously (13) and, inview of the relative sizes of the polypeptides found, dimeriza-tion is a possible explanation. Nevertheless the size of thesmaller subunit and the zinc content indicate a polypeptidesimilar to that found previously for parsley (Table IV). Theminimum specific zinc content of the Tradescantia carbonicanhydrase was intermediate between the molecular weightestimates of the native and denatured enzymes (Table IV), andso it is difficult to say whether there is one zinc atom perpolypeptide or per native enzyme molecule. The absence of asmaller molecule (around 14,000) on the SDS gels likewisedoes not allow for a conslusion about the number of poly-peptides in the native carbonic anhydrase.

The presence of two distinct (electrophoretically resolved)enzymes in purified preparations, together with the fact thatthey appear not to be freely interconvertible lends at least somesupport to the idea of carbonic anhydrase isoenzymes in theleaves of higher plants (2). While for the Pisumn enzyme prepa-ration the maximum protein and maximum activity werecoincident (Fig. 2B), this was not the case for the purifiedTradescantia enzyme (Fig. 2A). A similar result was foundin preparative disc gel electrophoresis of the purified parsleyenzyme by Tobin (see Fig. 2 in ref. 16) and might be explainedby the presence of two carbonic anhydrases differing in theirspecific activity.

As has been observed previously (3-5, 15) the sulfonamideswere about 1000 times less inhibitory to plant carbonicanhydrases than to the erythrocyte enzymes. The reverse wasfound for inorganic anions, nitrate being 500 times more effec-tive against the Pisuml enzyme (Table III) and 50 times moreeffective against the enzyme in spinach leaf extracts (4) thanagainst human erythrocyte carbonic anhydrase (17). Themarked differences in sensitivity to these inhibitors shown bythe plant and animal enzymes suggest that the nature of andrelationship between the active and inhibitor-binding sitesmay not be the same in each case.The lack of inhibition by other anions such as sulfate and

phosphate and a much lower sensitivity to chloride (4) suggeststhat the endogenous levels of free nitrate or nitrite (4) ionmight regulate carbonic anhydrase activity in leaves.

If. as suggested by the behavior of the enzyme fromTradescantia, the "monocotyledon" carbonic anhydrases aremore labile in solution than those from dicotyledons, thensome of the difference in levels observed previously (2) mightbe explained by a difference in stability following extraction.Waygood and Clendenning (19) found a rapid (half-life at pH9 about 2 hr) inactivation of partially purified carbonicanhydrase from Tetr-agonia expansa with an optimum forstability at pH 6. This study, however, was done in the absenceof a reducing agent, and as found previously for the parsleyenzyme (1 6), both the enzymes isolated were more rapidlyinactivated in the absence of mercaptoethanol at all the pHvalues tested.

Acknowledgments-The authors are grateful to Dr. J. Wilmshurst, CSIRO Di-viSion of MAineralogy, for the utse of an atomic absorption spectrometer and toMr. M. B. Smith and Miss J. F. Back, CSIRO Division of Food Research, for asample of purified ovalbumin ancl for the determination of sedimentation veloc -itx. The technical assistance of Mr. D. Hockley is also gratefully ackno-wledged.

222 Plant Physiol. Vol. 50, 1972




LITERATURE CITED

1. ANDREWS, P. 1965. The gel-filtration behavior of proteins related to theirmolecular weight over a wide range. Biochem. J. 96: 595-606.

2. ATKINS, C. A., B. D. PATTERSON, AND D. GRAHAM. 1972. Plant carbonic an-

hydrases. I. Distribution among species. Plant Physiol. 50: 214-217.3. BRADFIELD, J. R. G. 1947. Plant carbonic anhydrase. Nature 159: 467-468.4. EVERSON, R. G. 1970. Carbonic anhydrase and CO2 fixation in isolated chloro-

plasts. Phytochemistry 9: 25-32.5. FELLNER, S. K. 1963. Zinc-free plant carbonic anhydrase; lack of inhibition

by sulfonamides. Biochim. Biophys. Acta 77: 155-156.6. KONDO, K., T. YONEZAWA, AND H. CHIBA. 1952. Plant carbonic anhydrase. I.

Confirmation of plant carbonic anhydrase and preliminary isolation. Bull.Res. Inst. Food Sci., Kyoto Univ. 8: 1-16.

7. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951. Pro-tein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.

8. MAREN, T. H. 1967. Carbonic anhydrase: chemistry, physiology, and inhibi-tion. Physiol. Rev. 47: 595-781.

9. MIARGOLIS, J. AND K. G. KENRICK. 1968. Polyacrylamide gel electrophoresis ina continuous molecular sieve gradient. Anal. Biochem. 25: 347-362.

10. PATTERSON, B. D., C. A. ATKINs, D. GRAHAM, AND R. B. H. WILLS. 1971.Carbonic anhydrase: a new method of detection on polyacrylamide gels usinglow temperature fluorescence. Anal. Biochem. 44: 388-391.

11. RicKLi, E. E., S. A. S. GHAZANFAR, B. H. GIBBONS, AND J. T. EDSALL. 1964.Carbonic anhydrases from human erythrocytes. Preparation and propertiesof two enzymes. J. Biol. Chem. 239: 1065-1078.

223

12. Rossi, C., A. CHERSI, AND M. CORTIVO. 1969. Studies on carbonic anhydrasefrom spinach leaves: isolation and properties. In: R. E. Forster, J. T.Edsall, A. B. Otis and F. J. W. Roughton, eds., C02: Chemical, Biochemicaland Physiological Aspects. National Aeronautics and Space Administration,Washington, D.C. pp. 131-138.

13. SHAPIRO, A. L., E. VINrELA, AND J. V. MAIZEL JR. 1967. LIolecular weight esti-mation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.Biochem. Biophys. Res. Commun. 28: 815-820.

14. SIBLY, P. M. AND J. G. WOOD. 1951. The nature of carbonic anhydrase fromplant sources. Aust. J. Sci. Res. Ser. B 4: 500-510.

15. TOBIa-, A. J. 1969. Purification of carbonic anhydrase from parsely leaves. In:R. E. Forster, J. T. Edsall, A. B. Otis and F. J. W. Roughton, eds., C02:Chemical, Biochemical and Physiological Aspects. National Aeronautics andSpace Administration, Washington, D.C. pp. 139-140.

16. TOBIa, A. J. 1970. Carbonic anhydrase from parsely leaves. J. Biol. Chem. 245:2656-2666.

17. VERPOORTE, J. A., S. MEHTA, AND J. T. EDSALL. 1967. Esterase activities ofhuman carbonic anhydrases B and C. J. Biol. Chem. 242: 4221-4229.

18. WAYGOOD, E. R. 1955. Carbonic anhydrase (plant and animal). In: S. P.Colowick and N. 0. Kaplan, eds., Methods in Enzymology, Vol. 2. AcademicPress, New York. pp. 830846.

19. WAYGOOD, E. R. AND K. A. CLENDENNING. 1950. Carbonic anhydrase in greenplants. Can. J. Res. C. 28: 673-89.

20. WILBUR, K. M. AND N. G. ANDERSON. 1948. Electrometric and colorimetric de-termination of carbonic anhydrase. J. Biol. Chem. 176: 147-154.

Plant Physiol. Vol. 50, 1972



CORRECTIONSVol. 50: 218-223. 1972.Atkins, C. A., B. D. Patterson, and D. Graham. Plant Car-

bonic Ahnydrases. II. Preparation and Some Properties ofMonocotyledon and Dicotyledon Enzyme Types.

Page 219, column 1, line 21, should be corrected to read:... and more ammonium sulfate (0.1 g to each ml) was ...

Page 221, Table III. The concentrations of acetazolamide andethoxyzolamide are too high by a factor of 10. They shouldbe corrected to read: 45.0, 27.0, 0.5, and 1.8, respectively.

Vol. 50: 347-354. 1972.Hansen, J. B., B. L. Bertagnolli, and W. D. Shepherd. Phos-

phate-induced Stimulation of Acceptorless Respiration inCorn Mitochondria.

Page 351, column 1, last paragrph, line 8, should be correctedto read: ... formation is 7 to 8 nmoles ATP/sec mg pro-tein. This corre- . . .

Vol. 50: 790-791. 1972.Zobl, R., G. Fischbeck, F. Keydel, E. Latzko, and G. Sperk.

Complementation of Isolated Mitochondria from SeveralWheat Varieties.

90, column 2, the last sentence which continues on page791, column 1, the information should be corrected to read:The (nonsignificant) calculated values for complementationdid not exceed the mean of the parent varieties more than6.5%. Furthermore, no correlation with the results of fieldtrials with the F1 hybrids is indicated. This remains true forheterosis of kernel yield as well as for straw height.

Since the cross between the varieties Jubilar and Diplomatshowed a very pronounced and significant heterosis effect inkernel yield, we have used isolated mitochondria from thesevarieties for biochemical investigations as reported by Sar-kissian (4) and Sarkissian and Srivastava (6). According toTable II the 1:1 mixture of mitochondria from these varie-ties were ...

Vol. 51: 727-738. 1973.King, R. W. and Jan A. D. Zeevaart. Floral Stimulus Move-

ment in Perilla and Flower Inhibition Caused by Nonin-duced Leaves.

Page 736, Figure 7 legend, should be corrected to read: Serialsections taken through internodes 1, 2, 3, and 4 ...

301

plant carbonic anh-vdrases · 2020. 1. 18. · stability in solution, and sensitivity to...

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