A gel-scanning method for kinetic studies on an acetylcholinesterase isozyme

ANALYTICAL

45, 486487

HICCHEMISTHY

A Gel-Scanning on

(1972)

Method

for

Kinetic

an Acetylcholinesterase

Y. C. CHIU, R. K. TRIPATHI, Section of Neurobiology

and

Behavior, Received

Cornell June

Studies

lsozyme AND R. D. O’BRIEN University,

Ithaca, New York 14550

1, 1971

There are a number of methods, calorimetric (l-3)) titrimetric (4), fluorometric (5)) radiometric (6), manometric (7), which have been used for quantitative assay of cholinesterase (ChE) activity in aqueous systems. Recently the multiple forms of ChE from various sources have become of great interest, and are studied most readily by gel electrophoresis. Several histochemical techniques have been adapted for qualitative demonstration of ChE isozymes on gels. They include the method of Karnovsky and Roots (8) using thiocholine esters as substrates, and the rather nonspecific method of Gomori (9), modified by Tripathi and Dixon (lo), using a-naphthyl acetate to determine the esterase activity in the gel. We have attempted to modify and extend these methods for quantitative studies on gels, so that, after automated gel scanning, one can establish kinetic constants for various substrate and inhibitors. The present report primarily deals with the experimental procedures and the nature of the kinetic parameters that were obtained on an acetylcholinesterase isozyme. MATERIALS

AND

METHODS

Enzyme. Bovine erythrocyte acetylcholinesterase (AChE) from Winthrop Laboratories was used. The enzyme solution was freshly prepared in phosphate buffer (10 mM, pH 7.0) containing 20% sucrose; 50 ~1 of this enzyme solution containing 40 pg of enzyme was applied to each gel. The specific enzyme activity was 1.2 pmoles/min/mg enzyme when assayed with acetylcholine by a pH-stat method at 25”C, pH 7.0. Electrophoresis. Polyacrylamide gel electrophoresis was performed by the method of Davis (11) as modified by Eldefrawi et al. (12). The thiocholine method of Karnovsky and Roots (8) for histochemical study was essentially followed. In order to demonstrate that the present method can also be applied to other histochemical agents, such as 480 @ 1972

by

Academic

Press,

Inc.

GEL-SCANNING

ASSAY

O,F

AChE

ACTIVITY

481

,8-naphthyl acetate, indoxyl acetate and phenyl thioacetate, the nonspecific diaeo-coupling procedure of Gomori (9), modified by Tripathi and Dixon (lo), was also used for comparison. The gels after electrophoresis were equilibrated at 23°C for 10 min in the respective incubation buffer medium. For kinetics of catalysis, two gels each were then transferred into various concentrations of substrate medium with continuous shaking at 23” for 10 min with oc-naphthyl acetate (NA) or 15 min with acetylthiocholine (ATCh). For inhibition studies, two gels each were incubated in a beaker containing 50 ml of the desired concentration of inhibitor at 23”. After the indicated time interval, inhibition was terminated by transferring the gels into the desired substrat,e medium for color development of the residual enzyme activity. After incubation with substrate, the enzymic reaction was stopped by dipping t’he gels into 7% of acetic acid for fixation. The enzyme bands appeared as brown and dark purple for ATCh and NA respectively. The colors of the zymogram from both staining methods remained stable for hours in this fixative. They all exhibited a strong and wide adsorption band in the region of 400 rn+ Gels after fixation were therefore scanned arbitrarily at 410 rnp with a Beckman ACTA model recording spectrophotometer equipped with a gel scanner at 0.2 mm slit width. The enzyme activity expressed in terms of absorbancy was measured at the peak of absorbancy. In many cases even better results would be likely if one measured the area under the absorbancy curve, but isozymes commonly occur in groups with their bases overlapping, so we chose the present approach as being the most general. Determination of Michaelis consfant ik’,,,) and bimolecular rate constanfs (lci). The kinetic data. for catalysis were computerized by the weighted linear regression technique of Wilkinson (13) and plotted by the Lineweaver-Burk method. The kinetics of the inhibition reaction of the enzyme with various concentrations of inhibitors was expressed by plotting log % activity against time as described previously (14). The first-order rate constant (k) and its standard error were measured by a compmerized least-square procedure. Bimolecular rate constant iki) for inhibition were then obtained by the equation ki = k/i, where i was inhibitor concentrat,ion. RESULTS

The enzyme activity as a function of enzyme concentration at 5 x 1O-aM ATCh is shown in ‘Fig. 1. The absorbance increases with enzyme concentration up to about’ 40 pg, and then starts to level off. This is due to the fact, that, the amount of enzyme in earh gel significantly affects

482

CHIU,

TRIPATHI,

AND

O’BRIEN

40 Enzyme (pg) FIG.

with

1. AChE activity 15 min incubation.

as function

of enzyme

concentration

at 5 X 10” M

ATCh

the band width. When the amount of enzyme applied is more than 40 pg, there is a pronounced broadening of the enzyme band. This finding is in agreement with the recent observation in LDH isozymes (15) and in RNA electrophoresis (16). The rate of enzymic hydrolysis of ATCh and Na by AChE in the gel as a function of time is shown in Fig. 2. 10 and 15 min are the suitable lengths of time to develop an optimal range of absorbancy for NA and ATCh, respectively. However, with a very low enzyme activity, the incubation time can be arbitrarily prolonged to allow a sufficient color of measurement. Michaelis

Constants

(K,)

of ATCh

K,,, x Substrate ATCh cu-NA a Ratio

lo-’

-

of gel-scanning

0.8 + 0.9 method

with

for AChE

by Different

Methods

M Calorimetric

Titrimetric

7.9

TABLE 1 and (u-NA

f 0.1 -

calorimetric

Gel scanning 6.0 zk 0.4 37 2 f 12.3 or titrimetric

method.

K,,,

ratio0 7.5 4.7

GEL-SCANNING

ASSAY

Time FIG. 2. AChE activity of 40 pg AChE/gel: (0)

OF

AChE

483

ACTIVITY

(min)

as function of incubation 1 X 10m3 M wNA, (0)

time at enzyme 5 X 10e4 M ATCh.

concentration

The kinetic characteristics of AChE as determined by the doublereciprocal plot for both ATCh and NA are shown in Fig. 3. The K, values for ATCh and NA were 0.6 mM and 3.7 m&I, respectively. These values are about 5- to S-fold larger than those obtained in solution by calorimetric or titrimetric method (Table 1). The kinetics of inhibitions with Tetram (an oxalate salt of O,Odiethyl S- (2-diethylaminoethyl) phosphorot,hiolate) and malaoxon (O,O-

FIG. 3. Lineweaver-Burk (Y-NA

with

5 min

incubation,

plot

of ATCh (0) ATCh

and wNA with 40 pg with 15 min incubation.

AChE/gel:

(0)

484

A 0

CHIU,

TRIPATHI,

2.0

I .o

AND

~ 1.8 C .z t 1.6 0

Pi+ I

5

I

IO Time

O’BRIEN

se

I4

E -I 1.2 1.0

I

I

I

I5 (min)

20

5

I

IO Time

I

I5 (min)

I

20

FIG. 4. Log activity as function of time for enzyme inhibition with Tetram and malaoxon. Residual enzyme activity after inhibition for various times was assayed by 5 X lOA M ATCh (A) and 1 X 10e8M PNA (B), respectively. (0) (0) 1 X lo-“M malaoxon, (m) 2 X lCVJ4 Tetram, (0) 5 X lo-’ M Tetram, 2 X 1O-5 M malaoxon.

diethyl S- (1,2-dicarbethoxy) ethylphosphorothiolate) demonstrate that the inhibition rate displayed first-order kinetics as examined by both substrates (Fig. 4). The ICCvalues determined from both substrates were essentially identiCa1 (Table 2). The ki values were 5- to 11-fold lower than the values from the titrimetric method in an aqueous system for malaoxon and Tetram, respectively (17). This decrease of inhibitory power here is very similar in magnitude to the increase in K, for substrates.

Bimolecular

Rate

Constants

TABLE 2 (kc) of AChE 10-5

with

X ki (M-l

Tetram

and Malaoxon

mi0)

Substrate

Tetram

RIalaoxon

ATCh wNA

2.6 2.4 2.5a 28.0”

0.19 0.24 0.22a 1.12b

ACh

d Average k; obtained from the gel-scanning method with ATCh and o(-NA substrates. b Fstimated lci values from reference 18, assuming &II, = 2, obtained from titrimetric method using ACh as substrate.

as

GEL-SCANNIKG

ASSAY

OF

TABLE of Gel Concentration

Effect Gel concn.

(%I

3 on K,

104 x K,

7 9 11

6.9 8.0 S.l

AChE

485

ACTIVITY

and Relative

@I)

V,., min)

l’,,,(AOI>/l5

dz 1.0 + 1.4 + 0.6

2.8 3.3 3.8

* 0.2 * 0.3 * 0.2

The gel concentration might well have an effect sieving characteristics of a gel, thus affecting the ponents in the aqueous medium. An attempt was whether or not different gel concentrations would characteristics. Table 3 indicates that there was no

Effect

of Gel Concentration

TABLE 4 on Band Width of Enzyme Different ATCh Concentrations

on the porosity or diffusion of commade to examine affect the kinetic appreciable differ-

Expressed (mik’)

by Absorbance

with

01) Gel concn. (%)

~

7 9 11

0.2

0.5

1.0

0. .x3 0.6.59 0.773

1.222 1 ,342 1.426

1.616 1.886 2.103

ence in K, values with the three gel concentrations used. However, there was a pronounced increase in absorbance with increase of the gel concentration, thus affecting relative V,,,, significantly (Table 4). In order to establish the degree of variability, data were obtained from six runs of electrophoresis with two replicate gels each with TABLE of Sensitivity

Comparison

-

1OlX Method

Other

Methods

concn.

Amt.

of enzyme

W)

Calorimetric Calorimetric Titrimetric Gel-scanning Gel-scanning a 1 division = 0.025 was used as titrant.

substrate

5 with

ATCh IPA ACh ATCh ATCh wmole

ACh

hydrolyzed

5 5 30 3 5 at pH

b-d

Approximate OD/15 min

8 800 400 40 10

1.31 1.24 20 div./m& 1.25 0.90

7.0, 25”C,

when

6.25 mM

NaOH

486

CHIU,

TRIPATHI,

AND

O’BRIEN

ATCh as substrate. The mean OD was 1.253 with a standard deviation of 0.051, which is 4.1%. There is therefore excellent reproducibility. A comparison was made of the sensitivity of the present method with other procedures widely used for assaying AChE (Table 5). The data indicate the gel-scanning method was quite comparable with the colorimetric method of Ellman (3)) which provided one of the most sensitive and inexpensive ways of assaying this enzyme. DISCUSSION

There are several advantages for this gel-scanning technique. Kinetics of enzyme catalysis and inhibition can be performed directly on the gel. Kinetics of several different forms of isozyme can be conveniently resolved from a single zymogram; there is no need for laborious elution of separated isozyme from a preparative column. Low enzyme activity can also be resolved by prolonging time with substrate. Good reproducibility and high sensitivity provide a great asset for assaying the soluble enzymes from different sources or various species of organism at the same time. The same techniques can be applied to assay many other esterases by using various nonspecific substrates, such as cynaphthyl acetate, ,B-naphthyl acetate, and indoxyl acetate, which have been used in histochemical studies. Values of Km are increased and of Ici are decreased when one compares enzyme in the gel with that in solution. This difference is probably not due to an additional diffusional step in the gel, since the K, value does not vary with gel concentration. It may reflect the altered ionic medium within the gel ; it is known that the K, is very sensitive to salt concentration (18). In addition, several studies have indicated changes in kinetic parameters in bound enzyme as compared with free, including changes in K, of up to 50-fold (19-21). The method is therefore at its most useful when comparing kinetic properties of various isozymes on gels, especially when there are several isozymes on a single gel. SUMMARY

A gel-scanning technique involving gel staining and spectrophotometric scanning has been developed for quantitative assay of cholinesterase and other esterase isozymes. The kinetics of catalysis and inhibition can be directly determined without elution from the gel. ACKNOWLEDGMENTS GM

This research was 07804, Training We wish to thank

supported in part by funds from U. S. Public Health Grant E.S.-98, and a grant from Ford Foundation. S. Rhine and Y. K. Chang for their help in computer

Service analysis.

GEL-SCANNISG

ASSAY

OF

iiC1-lE

ACTIVITY

487

REFERENCES 1. HESTRIN, S., J. Biol. Chem. 180, 249 (1949). 2. KRAMER, D. N., AND GAMSON, R. M., AnaZ. Chem. 30, 251 (1958). 3. ELLMAN, G. L., COURTNEY, K. D., ANDRES, V., JR., AND FEATHERSON, R. M., Biochem. Pharmacol. 7, 88 (1961). 4. MAIN, A. R., AND IVERSON, F., Biochem. J. 100, 525 (1966). 5. KRAMER, D. N., AND GUILBAULT, G. G., Anal. Chem. 36, 1662 (1964). 6. WINTERINGHAM, F. P. W., AND DISNEY, R. W., Nature 195, 1303 (1962). 7. MENGLE, D. C., AND CASIDA, J. E., J. Agr. Food Chem. 8, 431 (1960). 8. KARNOVSKY, M. J., AND ROOTS, L., J. Hktochem. Cytochem. 12, 219 (1964). 9. GOMORI, G., J. Lab. Clin. Med. 42, 445 (1953). 10. TRIPATHI, R. K., AND DIXON, S. E., Can. J. Zool. 46, 1013 (1968). 11. Dam. B. L., Anx. N. E’. Acrid. Sci. 121, 404 (1964). 12. ELDEFRAWI, M. E., TRIPATHI, R. K., AND O’BRIEN, R. D., Biochem. Biophys. Acta 212, 308 (1970). 13. WILKINSON, G. N., Biochem. J., 80, 324 (1961). 14. ALDRIDGE, W. N., Biochem. J. 46, 451 (1950). 15. FRITZ, P. J., MORRISON, W. J., WHITE, E. E., AND VESSALL, E. S., Anal. Biochem. 36, 443 (1970). 16. RICHARDS, E. G., AND LECANIDOU, R., Anal. Biochem. 40, 43 (1971). 17. CHIU, Y. C., AND DAUTERMAN, W. C., Biochem. Pharmacol. 19, 1856 (1970). 18. ROUFOGALIS, B. D., AND THOMAS, J., Life Sci. 7, 985 (1968). 19. PENNINGTON, S. N., BROWN, H.. D., PATEL, A. B., AND KNOWLES, C. O., Biochim. Biophus. Acta 167, 479 (1968) 20. WEBB, G. D., AND HOHNSON, R. L., Biochem. Pharmacol. 18, 2153 (1969). 21. SILMAX, I., in “Proceedings of a Symposium on Membrane Protein.” pp. 50-57. Little, Brown, Boston, 1969.