carbonic anhydrase of sepia officinalis l

Comp. Biochera. Physiol., 1971. Vol, 38B, pp. 707 to 721. Pergamon Press. Printed in Great Britain

CARBONIC ANHYDRASE OF SEPIA OFFICINALIS L.

A. D. F. A D D I N K

Laboratory for Comparative Physiology, Department of Chemical Animal Physiology State University of Utrecht, The Netherlands

(Received 28 August 1970)

Abstract - -1 . Carbonic anhydrase of the mantle muscle of the cephalopod, Sepia offia'nalis, is a zinc protein, comparable to the highly active soluble vertebrate isoenzyme present in human and bovine red blood cells.

2. A procedure for the extraction of the membrane bound enzyme using detergents, acetone and centrifugation is presented, resulting in a purification of more than forty-five times.

3. Kinetic parameters of the CO2-hydration activity of the enzyme are determined by a stopped-flow spectrophotometric method and found comparable to the highly active vertebrate isoenzyme.

4. The inhibition by sulfonamides is established. The dissociation constants for the acetazolamide and ethoxzolamide ,Sepia carbonic anhydrase complexes are determined.

5. Electrophoretic behaviour of the invertebrate enzyme is also compared with vertebrate enzymes.

INTRODUCTION IN 1937 Ferguson et al. established the presence of a carbonic anhydrase (carbonate hydrolyase E.C. 4.2.1.1) in the gills and other tissues of the mollusc, Loligo pealeii, as determined by the catalyzed conversion of bicarbonate to COy The activity of this enzyme in several tissues, including mantle muscle of Sepia offidnalis has been published in a review by Van Goor (1948). The blood of Cephalopoda in contrast to blood of vertebrates does not contain any carbonic anhydrase activity. Physiological experiments concerning the role of carbonic anhydrase in muscles of cephalopods are not known, although an influence of the pCO 2 or changing pH on the respiratory activity has been noted (Winterstein, 1925; Wilbur & Yonge, 1964). Analogous to the mammalian carotid bodies controlling ventilation, a receptor sensitive to increased pCOz or acid levels may be proposed in the molluscan system (Lee & Mattenheimer, 1964).

The electron microscopic examination of the mantle muscle of S. officinalis (Addink, 1968) using glutaraldehyde as a fixative confirmed in general the morphology--"double-oblique striated muscle fibers with an interrupted Z-line, so-called J-particles"--as previously observed by Kawaguti & Ikemoto (1957) for S. esculenta and by Hanson & Lowy (1960) for Loligo. The physiological role of the zinc-metalloprotein carbonic anhydrase in vertebrate organ systems and the effects of inhibition as well as the chemistry of the processes involved

707

708 A . D . F . ADDINK

have been described by Maren (1967a) in an excellent and penetrating review. Also in 1967(b) Maren presented the phylogeny of carbonic anhydrase in red cell and kidney of vertebrates: throughout the vertebrate kingdom the chemical properties of the enzyme are very similar al though not identical. I t is of phylogenetic interest to study the characteristics of an invertebrate enzyme. In the present communicat ion the properties of carbonic anhydrase present in the mantle muscle of the molusc Sepia o~icinalis are compared with those of the enzymes from human and bovine red blood cells.

MATERIALS AND METHODS* Analytical grade chemicals were used throughout. Solutions were made in distilled

water, which was redistilled in a Jena glass apparatus with a quartz heating device. When it was necessary freshly boiled twice-distilled water was used.

The carbon dioxide solutions were prepared by bubbling mixtures of COs and N2 gas into water through a Jena glass fritted disk in a polyethylene bottle, thermostated at 25+0.1°C. The flow of the gases was controlled by two Flowmeters (Brooks Instr., Veenendaal, Holland). The concentration of COs was checked manometrically with the Natelson microgasometer, p-Nitrophenol (British Drug Houses) was recrystallized four times from water. The molar extinction (t) coefficients of the basic (In) and acid (HIn) forms of the indicator were determined at 400 m/z: em= 18,400 and emn = 160. The pK value of p-nitrophenol is 7"149 at 25°C (Robinson & Biggs, 1955). Acetazolamide, obtained from Lederle (American Cyanamid Company, U.S.A.) as Diamox, was used without further purification. It contained 1"6 equivalent sodium for a ready solubility. Ethoxzolamide was a gift from Upjohn Company (U.S.A.). The inhibitor was extracted from the 125 mg tablets. It was four times recrystallized from ethylacetate-hexane before the pure white powder was used. The living animals were obtained from the Nederlands Instituut voor Onderzoek der Zee at Den Helder, the Netherlands. They were caught by fishermen in the North Sea in summer time. No selection of male or female, younger or older animals was made. The weight varied from 1"6 to 4 lb. The pieces of isolated tissue of decapitated animals were stored at - 27°C.

Two assay systems were used to determine the activity of carbonic anhydrase in the hydration of COs, both based on a pH indicator system. For the crude tissue homogenates and turbid enzyme preparations an optical bromothymol blue system analogous to the method of Roughton & Booth (1946) was used as described below.

For the more purified enzyme preparations a stopped-flow system was used. A simplified stopped-flow apparatus adapted to a Beckman DU spectrophotometer has been described by Stewart & Ouellet (1959); this apparatus has been modified and fitted to the Beckman double-beam spectrometer. A complete description and diagram has been given elsewhere (Addink, 1968). The change of transmittance of the assay mixture containing p-nitrophenol in the flow-through cell is recorded by means of a Sargent SRL recorder at a chart speed of 10 in/min. The spectrophotometer and the recorder were operating in an unmodified version. The mean mixing time of each 4"6 ml of the CO~-HzO solution and the enzyme phosphate-buffer-indicator solution with or without inhibitor was 0"52 see, corresponding

* Abbreviations used--Acetazolamide; 2 acetylamino-l,3,4-thiadiazole-5-sulfonamide. BCA (A or B): bovine carbonic anhydrase with the isoenzymes BCA-A and BCA-B. BTB unit: bromothymol blue unit; unit of carbonic anhydrase: the amount of enzyme that doubles the hydration velocity in the bromothymol blue system. DOC: sodium deoxycholate. Ethoxzolamide: 6-ethoxy-benzothiazole-2-sulfonamide. HCA (A, B or C) : human carbonic anhydrase, with the isoenzymes HCA-A, HCA-B and HCA-C. pCOz: partial pressure of COs. SCA: carbonic anhydrase of Sepia o~cinalis.

CARBONIC ANHYDRASE OF SEPIA OFFICINAL1S L. 709

with a flow of 17"7 ml/sec or over a liter/rain. The standard error is 0"8 per cent. The complete operation was done at 25"0_+0"1°C by means of a constant-temperature bath. The whole flow system was kept continuously full of fluid, reactants or water. The reference cell in the spectrophotometer was filled at each pH value with a special acid solution of p-nitrophenol (KH2PO4, p H < 5). The concentrations of the indicator and phosphate corresponded to that of the sample cell during a reaction, see Table 1. Each of the phosphate buffer-p-nitrophenol mixtures was calibrated with 0-1 N HC1. The changes in

TABLE 1 - - C O M P O S I T I O N OF THE UTILIZED REACTION MIXTURES IN THE

STOPPED-FLOW EXPERIMENTS *

Total phosphate Indicator Added enzyme concentration HPO~ ~- concentration preparation

pH (M) (M) Ionic strength (M) mg protein/100 ml

7-55 0.025 0"020 0-066 5"0 x 10 -~ 0.40 7.00 0.025 0"013 0"052 7"5 x 10 -6 0"60 6.42 0-025 0"006 0"037 1"5 x 10 -6 0"75

* At each pH value three concentrations of CO2 are utilized: 4"2, 7"8 and 12"6 raM.

optical density were found to be proportional to the concentration of HC1 up to 5 mM corresponding to a pH change of about 0.4 units.

Kinetics of the hydration of CO2, uncatalyzed, catalyzed and after inhibition

The technique with the stopped-flow apparatus in the present study is similar to that of Kernohan et al. (1963), Ho & Sturtevant (1963) and Gibbons & Edsall (1963). The reaction was initiated by rapid mixing of 4"6 ml aqueous solution of CO2 and 4.6 ml phosphate buffer solution containing indicator for measurements of the uncatalyzed reaction. The velocity of the catalyzed reaction was determined by addition of the enzyme preparation to the phosphate-buffer-indicator mixture before the rapid mixing. Measure- ments with inhibitors were made after addition of the inhibitor to the enzyme-buffer- indicator mixture, at least 60 min before the rapid mixing with the COz solution. As equilibrium between, for example, acetazolamide or ethoxzolamide and carbonic anhydrase is reached in 2 rain (Maren, 1967a) a change due to the association phenomena is not expected. The dissociation of the enzyme-inhibitor complex requires several days (see below). So during measurements no significant alterations would be expected to result from the 1 : 1 dilution in the rapid mixing procedure. The indicator is virtually non- inhibitory at the concentrations used. For each particular pH value and CO~ concentration a series of seven measurements was performed. The mean of such a series appeared to have a standard error ranging from 2 to 5 per cent.

The velocity constant for the tmcatalyzed hydration reaction was determined by measuring the initial rate as a function of the CO2 concentration at pH 7"55. The uncatalyzed reaction at pH < 8 occurs in two ways to produce bicarbonate, analogous to the hydration scheme of SO~ of Eigen et al. (1961):

H + + HCOs- -----~, H2CO3

H20 + CO~

710 A . D . F . ~2)DINK

The actual value for the total uncatalyzed hydration velocity of CO, was determined to be 0"0377 sec -1, a mean value for seven measurements (Addink, 1968). This value is in agreement with those found by Kernohan (1965) and Gibbons & Edsall (1963): 0.375 and 0"0386 sec -1 respectively.

The velocity or initial rate of the enzyme-catalyzed hydration reaction (v,) was obtained from the difference between the total velocity (v~) and the velocity in absence of the enzyme (v0):

The Michaelis constant K,~ and maximal velocity Vmaz of the catalyzed hydration were determined from Lineweaver-Burk plots in the usual manner (Dixon & Webb, 1964). The dissociation constant of the enzyme-inhibitor complex K~ was also determined. The actual enzyme concentration E0 in a semi-purified preparation was determined by means of the formation of an enzyme-inhibitor complex.

E + I . • EI.

Maren et al. (1960) have investigated the inhibition of carbonic anhydrase by several inhibitors in the manner suggested by Easson & Stedman (1936). As the sulfonamides form a carbonic anhydrase-inhibitor complex at a 1 : 1 ratio (Lindskog, 1963) it is possible to obtain E0 by titrating an enzyme preparation with the aid of a highly potent inhibitor, e.g. ethoxzolamide (Kernohan, 1965). The equations, which are usually expressed in terms of free inhibitor, require modifications because the concentration of free inhibitor is greatly reduced. Assuming reversibility and noncompetition (see Results) the equation developed by Easson and Stedman is

I0 K~ l l_~i + Eo , i

in which I0 is the original concentration of inhibitor, i is the fractional inhibition and Ks is the dissociation constant of the enzyme-inhibitor complex. Plotting these data it is possible to calculate E0.

Bromothymol blue method The method was taken from that of Roughton & Booth (1946) according to instructions

kindly given by Dr. C. A. Mawson of the Biology Branch of Atomic Energy Ltd. of Canada in Chalk River, Ontario. Our modification of the method consisted of the comparison of the hydration velocity of a carbonic anhydrase catalyzed reaction with that of the completely inhibited enzyme reaction in order to correct for a possibly changed buffer capacity due to addition of noncatalytic proteins.

The assay mixture consisted of 3 ml veronal buffer, pH 7.95 (0"022 M sodium diethyl- barbiturate and 0"022 M diethylbarbituric acid), 0"1 ml bromothymol blue solution (0.04 g/100 ml diluted with water from a 0.01 N NaOH solution), and 2 ml peptone water (0.25% peptone made up in CO,-free water to stabilize dilute enzyme solutions), 0"3 ml enzyme solution and 0.1 ml water or inhibitor.

All materials used were reagent grade except bromothymol blue (Merck indicator), peptone (British Drug Houses) and the sulfonamide inhibitor acetazolamide (Lederle). For each estimation two stoppered 15-ml weighing bottles, with a lid coming down over the outside of the bottle, were used (Scitec, Amsterdam). One weighing bottle as optical comparison tube contained 0-1 ml indicator and 10"4 ml phosphate-citrate buffer, p H 6"30 (0"135 M NaaHPO4+ 0"032 M citric acid). To check for the uncatalyzed CO2-hydration reaction two weighing bottles (C) were included containing 0.4 ml water. These control bottles with water were labeUed C, the sample bottles 1, 2, 3 and the bottles with inhibited enzyme D. The measurement was made in the following order: C, 1, 1D, 2, 2D, 3, 3D, 2, 2D, 1, 1D, C. The bottles (D) contain the above mixture but, instead of 0"1 ml water,

CARBONIC A~IHYDRASE OF SEPIA OFFIC1NALIS L. 711

acetazolamide was added at a final concentration of 10 -~ M in order to inhibit the enzyme completely. The effect of the addition of the sodium salt of acetazolamide on the uncatalyzed reaction time was very small: it was increased by 1 sec for which a (constant) correction could be made. The whole series of bottles was put in the cold room ( + 1 °C) into a dish containing crushed ice. With the aid of a cooled graduated syringe 5 ml of saturated COs solution (0.07 M CO2) was introduced into the contents of the weighing bottles. The long polyvinylchloride nozzle (4 in.) was pushed to the bottom and the contents of the syringe were expelled violently to provide good mixing. After the addition a stopwatch was started and the cap of the bottle replaced. The mean time necessary for the uncatalyzed reaction was about 82.0 sec (control time). The units per bottle were calculated from the mean of the time in seconds required to reach pH 6.3 :

inhibited test t i m e - test time Units per bottle (BTB units) --

test time

In practice, t ime of the active samples was kept between 20 and 40 sec, if possible. No allowance could be made for the limiting effect of diffusion (Roughton & Clark, 1951; Waygood, 1955). The progressive inactivation of very dilute enzyme solutions was pre- vented by addition of peptone.

One BTB unit was calculated to be equivalent to about 5 x 10 -1~ M Sepia carbonic anhydrase as obtained from the molar concentration by the stopped-flow method under comparable condition except for the larger change of pH.

On addition of detergents the indicator bromothymol blue was solubilized, probably in the detergent micelles, thereby changing its color to yellow, at a constant p H (7.95). For instance, the critical micellar concentration of Tri ton X-100 is 0.01% by weight as indicated by the manufacturer, Rohm and Haas Comp., whereas the concentration effective in liberating the enzyme was 0.1%. Appropriate dilutions were made to reduce this effect.

Agar gel electrophoresis

A modified method of Wieme (1965) was followed. The 1% agar layer in a barbital buffer, p H 8"6, with an ionic strength of 0.025 was 3 mm thick on glass slides measuring (3 x 12 cm) provided with two Z-bent strips of paper (2.5 x 3 cm) Whatman No. 3, as agar-buffer bridges. In the covered plexiglas apparatus the electrode compartments were filled with barbital buffer, pH 8.6, and ionic strength of 0"050. A constant voltage of 120 V direct current was applied for 1"5 hr (about 8 V/cm). As the slides and the electrophoresis apparatus were placed in the cold room ( + 1 °C) no further measures were taken to cool the electrophoresis slide. After fixation for 30 min in ethanol-acetic acid-water mixture ( 7 0 : 5 : 2 5 v/v) dehydration and desalting was performed by immersion for 4 hr and changing the fluid for 20 hr in 90% acetone (v/v). The slides were dried in a 35 °C oven. Staining of protein bands was accomplished by immersion in 0"5% napthalene black 10B (G. T. Gurr , London) (w/v), and 5% acetic acid v]v for 30 rain. After nonfixed dye was eluted with 2% acetic acid the slides were dried again.

To detect carbonic anhydrase activity in a non-fixed electrophoresis slide the CO~- hydration method with bromothymol blue was used again. The slide is immersed for 20 min in a BTB solution (0.4%) with or without 10 -8 M acetazolamide. After a short rinse with water the agar slide is placed beneath a large glass funnel (upside down). The funnel is connected to a CO2 gas cylinder. An equal distribution of the CO2 gas is effected by this means. The (blue) slide is observed carefully so as to record the emerging yellow spots (pH change). The sites of hydration of COl turn yellow faster than the background and were marked on the underlying white paper together with the time necessary to reach the yellow colour. The specificity of the catalytic hydration is checked with a parallel electrophoresis slide which was immersed in the indicator solution containing 10 -8 M acetszol- amide. The inhibited reaction should take a longer time than the catalyzed reaction. The


recorded periods were about 5-50 sec for the catalyzed reaction and 150-360 sec for the inhibited or uncatalyzed reaction. However, a spot with a high concentration of inactive protein turned yellow more rapidly than the background. Afterwards these slides were fixed and stained for protein. The minimal amount of protein which might be detected with the napthalene black 10B staining technique was found to be 1/zg bovine serum albumin, which corresponds nearly to that reported by Bennett & Boursnell (1962) as being 0.5/zg. The esterase activity of carbonic anhydrase (Pocker & Storm, 1968) was assayed for in agar electrophoresis slides with fl-naphthyl-acetate according to the method of Uriel, quoted by Wieme (1965). Inhibition of carbonic anhydrase esterase activity could be completely inhibited in 10 -3 M acetazolamide.

Zinc determination

The zinc content of tissues and carbonic anhydrase preparations was determined by the atomic absorption spectrophotometric method at the resonance absorption of radiation at 2138"6 A, Perkin-Elmer instrument, model 303. Tissues were destructed according to Kahnke (1966) in a HNO3-HCIOI mixture.

Results of the extraction procedure of carbonic anhydrase from mantle muscle of S. officinalis

The final simplified large-scale procedure consisted of a combination of the methods found favourable and not time consuming (Addink, 1968). AU treatments were made at +1°C. Sepia mantle muscle (or gill) was prepared by removing skin and attached connective tissue. This tissue was diced and after addition of two volumes of 0.01 M K2HPO4, 0.1% Tri ton X-100 and 0"1~o toluene, blended in the Waring Blendor with the anti-frothing device four times for 30 sec. This mixture was centrifuged at 180 g for 10 min. The resulting sediment was re-extracted with one volume of the medium. The turbid supernatant was mixed with two volumes of reagent acetone ( -20°C) . The precipitate obtained after centrifugation at 6000 g for 7 rain was mixed with 6 vol. of acetone and centrifuged again. Tri ton X-100 and toluene are largely removed by this procedure. The resulting white precipitate was crumbled somewhat and rapidly dried in a vacuum desiccator over P205. The resulting powder could not be dissolved in aqueous buffer solutions. The material contained 210 mg protein/g and the carbonic anhydrase activity was 30 BTB units per mg protein. To solubilize the carbonic anhydrase, an amount of 40 mg was mixed with 20 ml H20 containing deoxycholate (DOC), the ratio of DOC to protein being 1"6. After centrifugation at 49,500 g for 15 min the clear supernatant was utilized for the kinetic measurements. In this way a purification of the enzyme of forty-seven times was obtained (Table 2).

TABLE 2 - - I N C R E A S E IN ACTIVITY OF THE Sepia CARBONIC ANHYDRASE PREPARATION

AS A RESULT OF THE EXTRACTION PROCEDURE

BTB units/mg protein

Sepia mantle muscle homogenate After Tri ton X-100, (NH4)2SO~ and K2HPO4 Acetone powder After solubilization by DOC

1.3 5.0

30 61

At 400 m/z as measured against distilled water in the Beckman DB spectrophotometer the optical density was found to be less than 0"002 for the concentrated enzyme solution (1-cm cuvette). Moreover, in the kinetic experiments this solution was diluted twenty to fifty times. The u.v. spectrum showed a maximum absorption of 276 m/z and a minimum at 257 m/~. The preparation of carbonic anhydrase from gill tissue showed a final activity

CARBONIC ANHYDRASE OF SEPIA OFFICINALIS L. 713

of 90 BTB units/rag protein, however, the overall purification was less than that of muscle carbonic anhydrase.

RESULTS The velocity of the enzymatic hydration reaction was proportional to the

concentration of the SCA enzyme extract over a sixfold range as shown in Fig. 1. Lineweaver and Burk plots for the spectrophotometric stopped flow measurements of the enzyme-catalyzed reaction yield a straight line at each of the three pH values used: 6.42, 7.00 and 7-55. Figure 2 illustrates the plot for pH 7.0. In Table 3 the values of the hydration parameters Vma x and K m are summarized.

0.7

0,6

E 0-5

~ o.4

0-3

0.2

0.1

0 .0 I I I I I I I O.I 0.2 0.3 0 .4 0.5 0.6 0.7

rng prote in / lO0 mL

0-8

FIG. 1. Hydration velocity (%) as function of the Sepia carbonic anhydrase concentration (rag protein of the mantle muscle preparation/100 ml) in 0.025

phosphate buffer, pH 7"55. The CO= concentration was 12"6 mM.

The values for the concentration of Sepia carbonic anhydrase (E0) are calculated from the inhibition data. The turnover numbers increase with increasing pH. One enzyme unit, as recommended by the Commission on Enzymes of the International Union of Biochemistry (1965), although revised later, was defined as the amount of enzyme that catalyzes the transformation of one micromole of substrate per minute (at 25°C), preferably under standard conditions. At pH 7.55 (probably not optimal) an enzyme unit was calculated from the value of the turnover number (Table 3) assuming a molecular weight of 30,000. Then one enzyme unit of SCA corresponded to 7-0 x 10 -7 mg protein and the specific activity of 1 mg protein was 1,420,000 units.

Riepe & Wang (1968) have reported a competitive role of several inhibitors including sulfonamides in the CO2-hydration reaction as determined by a method of difference infrared spectrum of CO2-enzyme complex.


50

2O

L

s S s J

~ S S

• s , o " ~

J I L O' I0 &O0 0"10 O' 20 0"50

/ toM-1 $

FIC. 2. Lineweaver-Burk plot for the hydration of CO9 catalyzed by Sepia carbonic anhydrase (mantle muscle preparation) at pH 7"00 (0) . The dashed lines represent the hydration in the presence of 2 x 10 -9 M (lower) and 4 x 10 -9 M (upper)

ethoxzolamide (noncompetitive inhibition).

T A B L E 3 - - H Y D R A T I O N PARAMETERS OF CARBONIC ANHYDRASE OF

Sepia MANTLE MUSCLE AT 2 5 ° C *

HCA-C Km Vmax Eo Vm~,x/ Eo Vm~x/ Eo

(M x 10 3) (M see -x x 10 s) (M x 10 °) (see -x x 10 -3) (see -x x 10 -z)

pH 7"55 pH 7"00 pH 6"42

6.29 0.71 1-0 710 (640) 10.44 0.72 1.5 480 (620) 3.31 0.19 1-9 100 (290, at pH 6.3)

* The values of E0 of pH 7"55 and 6"42 are calculated from the inhibition data with ethoxzolamide of those of pH 7-00. K,~ and Vraax are found from Lineweaver- Burk plots. The figures of the turnover numbers of human carbonic anhydrase C (HCA-C) are taken from Table I I I of Gibbons and Edsall (1964). The conditions are similar. The turnover numbers per minute are 4"3 x 107, and 2"9 x 107, and 0-6 x 10 ~ M/M at the respective pH values.

We determined the value of the dissociation constant of the enzyme- inhibitor complex (Ki) for ethoxzolamide and acetazolamide (Table 4). The inhibition of the hydration reaction appeared to be noncompetitive (Table 5 and Fig. 2). The reason for this result under conventional conditions is the stability of the (reversible) enzyme-sulfonamide complex. Experimentally it is difficult to decide that ethoxzolamide is competitive unless the substrate concentration can be increased to inconveniently high partial pressures of COs, since the dissociation constant of the bovine carbonic anhydrase-CO 2 complex is 12.5 x 10 -3 M (Lindskog, 1960, 1962; Kernohan, 1964), which is 105 times

CARBONIC ANHYDRA~ OF SEPIA OFFICINALLS L. 715

TABLE 4 K~ VALUES OF ETHOXZOLAMmE AND AC]gTAZOLAMIDE FOR S ~ ' a CARBONIC ANHYDRASE (SCA)~ DETERMINED FROM THE ENZYME-INHIBITOR

COMPLEX SYSTEM*

SCA HCA-C

Ethoxzolamide 1 x 10 -9 M 2 x 10 -9 M Acetazolamide 2"5 x 10 -9 M 20 x 10 -9 M

* The somewhat higher values for human carbonic anhydrase C are given by Maren (1967a). These are determined from a system of the enzyme-inhibitor complex in the presence of the substrate.

TABLE 5--NoN-COMPETITIVE INHIBITION OF THE CATALYZED HYDRATION REACTION BY ETHOXZOLAMIDE AT p H 7"00 AND 25°C

CO2 concentration % Activity after addition of ethoxzolamide, at a final (mM) molar concentration of

8 x 10 -9 4 x 10 -9 2 x 10 -9 0"0 4-2 14-6 26"6 42"5 100

12"6 15 "7 26" 5 43 "2 100

higher than that of the carbonic anhydrase ethoxzolamide complex K¢, being 2 x 10 -9 M for pure human red cell carbonic anhydrases B and C (Maren, 1967a).

Another similarity with respect to mammalian carbonic anhydrases is the pH sensitivity. Sepia carbonic anhydrase appeared to be completely inactivated below pH 5 and above pH 10 as measured by the BTB method.

From the molar concentration of carbonic anhydrase obtained by the stopped- flow method it was calculated that 1 BTB unit corresponded to 5 x 10 -l° M Sepia carbonic anhydrase when measured under comparable conditions except for the larger change of pH. This value was comparable with that obtained by the titration of enzyme with ethoxzolamide being 6 x 10 -l° M.

From activity measurements of Sepia carbonic anhydrase in the presence of several concentrations of ethoxzolamide ranging from 10 -~ to 12 x 10 -o M with this BTB method also a K¢ of I x 10 -9 was calculated. This plot, as presented in Fig. 3 according to Easson & Stedman (1936), yields an enzyme concentration (E0) of 2.6 x 10 -9 M/1. (intercept on the ordinate).

The reversibility of the inhibition by sulfonamides was determined by a dialysis experiment for twelve days. About 450 BTB units of SCA in 20 ml, 0.04 M veronal buffer pH 7.95, and 5 x 10 -4 M acetazolamide were dialyzed in cellophane tubing (Kalle, Germany) against 1700 ml 2 x 10 -4 M veronal buffer, pH 7.95, daily renewed. A parallel experiment was run to investigate the loss of activity in a preparation without inhibitor. In 12 days the decrease in activity appeared to be considerable: 43 per cent. After correction for this "spontaneous" inactivation a nearly complete recovery of the activity was established (Fig. 4).

716 A . D . F . ADOINX

o

S I I I I I I I I I I I I 2 3 4 5 6 7 8 9 I0

I

FIO. 3. The inhibition of Sepia carbonic anhydrase by ethoxzolamide added at a final concentration of 1, 2, 4, 6, 8, 10, and 12 x 10 -9 M (I0). The activity was measured with the aid of the bromothymol blue method. The values are plotted according to Easson & Stedman (1936). The fractional inhibition is indicated by i.

I00

9O

8O

7O

6O

~ 50

40

30

2O

I0

I G' 2 4 6 8 I0 12

Days

FIG. 4. The reversibility of the inhibition by acetazolamide after correction for the spontaneous inactivation. The reactivation is more than 95 per cent.

T o reach 50 per cent of the original activity (corrected) about 120 hr ( T 0 are necessary for the dissociation of half of the enzyme- inh ib i to r complex. Maren (1967a) has repor ted the T t of the acetazolamide complex of H C A - B and C to be 80 and about 500 hr respectively. Apar t f rom the reversibili ty it is not possible

CARBONIC ANHYDRASE OF SEPIA OFF1C1NALIS L. 717

to draw any conclusion about the specific activity of the enzyme concerned, as more potent inhibitors show values of T t of 500 hr for both HCA-B and HCA-C.

Zinc results The final semi-purified muscle carbonic anhydrase preparation contained

78 #g Zn/g protein. A value of 164/~g gram was found for the gill enzyme. As a control a purified bovine carbonic anhydrase B appeared to contain 2 mg/g.

To what extent the amount of zinc determined could be related to its possible function in this invertebrate carbonic anhydrase is shown by the following experiment of reversible dissociation of zinc (Lindskog & Malmstr6m, 1962). Muscle enzyme preparation (66 mg) was homogenized in 6.6 ml 0.05 M acetate buffer, pH 5-0, containing 10 -3 M 1,10-phenanthroline. It was dialyzed at I°C in a Visking dialysis bag against 200 ml of the same solution, which was stirred with a Teflon-clad bar. Analogous to this experiment a dialysis experiment with muscle SCA was set up against buffer, pH 5-0, without phenanthroline to check for spontaneous inactivation, which appeared to be considerable: about 50 per cent in 2 days (Table 6). The percentages given are corrected for the spontaneous inactivation. Half of the zinc dissociated from the Sepia carbonic anhydrase in

TABLE 6"---DIALYSIS EXPERIMENT OF SCA AT pH 5"0 AGAINST 10 -3 M i,10-PHENANTHROLINE

% Activity found

After 1 day After 2 days

Without added zinc 61 50 After addition of 2 x 10 -3 M Zn 91 103

2 days (half time) during dialysis against 1,10-phenanthroline. This period corresponds to the half times determined by Lindskog & Malmstr6m (1962) for BCA-B and by Lindskog & Nyman (1964) for HCA-B. For BCA-A and HCA-C from 5 to 10 days are necessary for dissociation.

Results of electrophoresis of SCA In Fig. 5, examples of several electropherograms of Sepia, human and bovine

carbonic anhydrase preparations are presented. The proteins were visualized after staining with naphthalene black 10B, except for zone 8 in slide 6 in which the concentration of protein was below 1 t~g. The patterns obtained with HCA are in accordance with the data of Laurent et al. (1965) and Rickli et al. (1964).

The esterase activity was detected by the fl-naphthyl acetate method. In the slides 1 and 2 of HCA the zones 4, 5 and 6 were found to be positive: red zones. After addition of 10 -3 M acetazolamide no esterase activity was found in these zones. In slides 3 and 4 of BCA the zones 3, 4 and 5 were stained red. No staining could be detected after addition of inhibitor. Identical results were obtained after the bromothymol blue method was applied to these agar slides.


HCA

HCA- C

olb. 1 2 3 4 5 6 dext. 1T

' I1111 1 ' 1 i ABe

S

C

BCA I 2 345 6 I

BlIIDm D I AIAB

BCA- B 5 I B

SCA 1 2 3 4 5 6 7

i!iiiiiiiiWill I I s

SCA liliJiiiiit 1 +

FIG. S. Agar gel electrophoresis of carbonic anhydrase preparations. Agar slide I shows the electropherogram of the ethanol-chloroform preparation of carbonic anhydrase of human red blood cells. The zones 4, 5 and 6 represent the three isoenzymes of HCA: A, B and C respectively as indicated below the zones. In slide 2 the zone of HCA-C is shown after the purification procedure (ethanol- chloroform, gelfiltration and DEAE-Sephadex chromatography). Slide 3 shows the electropherogram of the ethanol-chloroform preparation of bovine erythrocytes. The zones 4 and 5 correspond to BCA-A and B respectively, whereas zone 3 (A') also shows a carbonic anhydrase activity. The zone of slide 4 is the purified BCA-B isoenzyme (ethanol-chloroform treatment, gel-filtration, and DEAE- Sephadex chromatography). The electropherogram of slide 5 shows the proteins of the supematant of a 20,000 g Sepia muscle-Tri ton X-100 homogenate after precipitation with acetone. Zone 1 is very broad, enclosing zones 2 and 3. In slide 6 zone 8 represents the Sepia carbonic anhydrase of the SCA preparation of muscle and gill (Triton X-100, acetone and DOC). Zone 8 was invisible after staining with naphthalene black 10 B, as the protein concentration was below 1/~g. All other numbered zones were visualized after protein staining. The intensity of the colour is indicated approximately by the intensity of the black tone. Dext ran-- to check the electro-osmotic flow---and bovine serum albumin (Poviet, Amsterdam, the Netherlands) were used as markers. These are indicated by dashed lines, "dextr" , and "alb." , on slide 1. The starting line is indicated by S.

CARBONIC ANHYDRAS~ OF SEPIA O F F I C I N A L I S L. 719

Moreover, in slide 6 in zone 8 the SCA activity was visualized. The active spots of two slides with SCA were cut out and assayed with our BTB method after mixing with peptone and buffer; the activity was found to be 1.2 BTB units, corresponding to 6 x 10 -12 M Sepia carbonic anhydrase. No esterase activity could be detected in this SCA zone, probably because the concentration was too low.

DISCUSSION The data of Table 3 and the electrophoretic mobility (Fig. 5) indicate that

Sepia carbonic anhydrase belongs to the rapid type of isoenzyme, e.g. HCA-C with a turnover number of 640 x 103 sec -t or BCA-B with 1000 × 103 sec -t (Kernohan, 1965). Some experiments carried out with this stopped-flow method with a carbonic anhydrase preparation of the gill of Sepia (Triton X-100- acetone-DOC) revealed an activity similar to that of the mantle muscle at pH 7.55. Maren (1967b) has concluded from the kinetic properties and responses to inhibitors of several vertebrate carbonic anhydrases from fish to man, that the human red cell carbonic anhydrase C may be regarded as a prototype of carbonic anhydrases throughout the vertebrate subphylum. From the data presented above it is possible to extend Maren's statement to a more highly organized representative of the invertebrates: Sepia oJ~idnalis.

The presence of deoxycholate appeared to be of no influence on the specific activity of bovine carbonic anhydrase neither on the velocity of the uncatalyzed reaction, as measured by the stopped-flow method at pH 7-55. So this detergent has a solubilizing function on the partly latent Sepia carbonic anhydrase (probably membrane-bound). This phenomenon is not found for the mammalian red blood cell carbonic anhydrases, as these enzymes are very soluble after hemolysis.

After the extraction of Sepia carbonic anhydrase from muscle tissue outlined in the methods, further purification procedures using DEAE-ceUulose, DEAE- Sephadex A50, Sephadex G75 and hydroxylapatite were not successful as nearly no activity could be measured in the eluate, using methods communicated by Lindskog (1960), Nyman (1961), Lindskog & Nyman (1964), Rickli et al. (1964) and Duff & Coleman (1966). All these chromatographic systems did work for purification of the soluble HCA-A, -B and -C from crude ethanol-chloroform extracts of human blood as well as the separation of BCA-A and BCA-B from extracts of bovine blood as shown in electrophoretic patterns of Fig. 5. The reason might be that the Sepia enzyme again eluted in a latent form. This problem will be of future concern. The following treatments were checked in relation to the purification procedure, they were found to produce no gross inactivation: high speed blending, ultrasonic treatment, ammonium sulfate precipitation, acetone treatment, neutral and some anionic detergents.

Opposite to these methods negative results were obtained with freezing and thawing, butanol treatment, 1% digitonin treatment, chloroform-ethanol method as communicated earlier (Addink, 1968).


CONCLUSION

From these experiments concerning carbonic anhydrase of Sepia o~cinalis it may be concluded that this invertebrate metallo-enzyme is comparable to the vertebrate isoenzyme with the high activity in the following respects: the essen- tial role of zinc, the hydration activity, the inhibition by sulfonamide derivatives and the electrophoretic mobility. The different solubility properties point to a membrane bound enzyme.

Acknowledgements--The author wishes to thank Professors H. J. Vonk and D. I. Zandee for their encouragement, Dr. J. W. Huismans for the many helpful discussions on enzymo- logical problems, Dr. Jennie M. Smoly of the Institute for Enzyme Research, University of Wisconsin, Madison, for assistance in the preparation of the manuscript, and Miss Wil M. E. Bloemsma for expert technical assistance.

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Key Word Index--Carbonic anhydrase, Sepia, human, bovine; Mollusca; Cephalopoda; Sepia ojTicinalis; zinc-metalloprotein; sulfonamides, inhibitors of carbonic anhydrase; invertebrate muscle; electrophoretic mobility of carbonic anhydrase isoenzymes.

carbonic anhydrase of sepia officinalis l

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