Isoenzymes of elapid acetylcholinesterases

('omp Btoch,,m Ph.lsiol. 1977. Iol 56('. pp 193 to 197. Per~lamon Press Printed in Great Britain

ISOENZYMES OF ELAPID ACETYLCHOLINESTERASES SU-RAY LEE, JENEFER LIu LATTA AND WILLARD B. ELI.IO'FI" Department of Biochemistry, State University of New York at Buffido, Buffalo. NY 14214, US.A.

(Received 5 April 1976) Al~tract--l. Acetylcholinesterase activity.of Naja naja naja, Naja melanoleuca and Bungarus Jasciatus venoms is due to mixtures of isoenzymes. 2. Naja melanoleuca venom has two acetylcholinesterase isozymes and both Nuja naja naja and Bungarusfusciatus venoms have four acetylcholinesterase isozymes that are resolved by disc gel electrophoresis. 3. With isoclcctric focusing, Naja melwtoleuca venom was found to have eight acetylcholinestcrase isozymcs with pl values ranging from 4.2 to 5.2, while Bumlarus fiisciatus venom had ten isozymes with pl values from 4.3 to 5.3. 4. The results of disc gel electrophoresis and ion exchange chromatography indicate that these isozvmes differ in charge. 5. Gel permeation chromatography of acctylcholinestcrase of Nuja melanoleuca and Bungarus.lusciatus venoms indicated that the isozymes of each venom all have a common molecular weight.

INTRODUCTION

Baker Chemical Corp. Ampholyte solutions (40% w/v) for isoelectric focusing with buffering capacities in the pH The existence of an enzyme which catalyzes the hyrange 3.5 10 were obtained from LKB Produkter, Bromma drolysis of acetylcholine was first suggested by Dale 1, Sweden. Coomassie brilliant blue G-250 was from Serva (1914). The presence of acetylcholinesterase (ACE; EC Feinbiochemica, Heidelberg. fl-carbonaphthoxy choline 3.1.1.7) in snake venom was reported over 30 years iodide was from Nutritional Biochemicals Corp• Diazo blue B was from Sigma Chemical Co. All the other chemiago by lyengar et al. (1938) and G h o s h el al. (1939). cals were of "'Analytical Reagent Quality." Zeller (1949) corroborated the finding of ACE in most Disc gel electrophoresis was done in glass tubes venoms from elapidae and noted its absence from 30 venoms of viperidae. Augustinsson (1963), Mebs {0.7 x 8.5 cm) in a Shandon apparatus, using the Ornstein & Davis method (1964). The gel solution was mixed as (1970) and K u m a r et al. (1973) found different concenfollows: 15ml 20% cyanogum 41 solution, 20ml o[ trations of ACE in various elapid venoms. McLean 0.12 M, pit 8.6, Tris-glycinc buffer. 4.7 ml distilled water, et al. (1971)studied the homology of elapid esterases 0.15 ml N.N.N',.\"-tctramethyleth31encdiamine and 0.15 ml capable of hydrolyzing fl-carbonaphthoxy choline of freshly made 20'% ammonium persulfate. After mixing, iodide (BCNCI) in relation to mobility when electro- the solution was lx)urcd into the glass tubes whose lower phoresed in starch or disc gels. K u m a r & Elliott ends were sealed with parafilm. The electrode chambers (1973) purified the ACE from Bungarus fasciatus were filled with 003 M Tris-glycine buffer, pH 8.6. The electrophoresis was at 4mA per tube for 1 hr for Naja venom. Since the pioneering work of Markert & M611er melanoleuca venom and 2 hr for Nuja naja naja and Bun~lurus/asciarus venoms at room temperature. (1959) on lactic dehydrogenase, a large n u m b e r of The isoelectric focusing technique used was modified enzymes have been found to exist in multiple forms from Righetti & Drysdale (1971). Gelling solution was pre(Vessell, 1968). ACE from other sources, e.g. erythro- pared by mixing together 20ml 15% cyanogum solution, cyte, head of face flies, rat retina, was made up of 3 ml, pH 3.5 10 ampholine solution, 16.7 ml distilled water, more than one form (Main, 1969; Knowles & Arun- 0.15 ml ammonium persulfatc and 0•15 ml N.N,N',N'-tetramethylethylcnediaminc. The solution was then pipetted kar, 1969; Davis & Agranoff, 1969). In this paper we report the existence of multiple into glass tubes (0.6 x 10 cm) whose ends were sealed with nylon mesh bound to the glass tubes with rubber rings forms of ACE in three elapid venoms. to avoid the leakage of the gel from the tube during electrophoresis. When the gels had formed, the tubes were inserted into the Shandon apparatus and the electrophoresis MATERIALS AND METHODS was done at 4"C. The electrode vessels were filled with Naja melanoleuca. Naja naja naja and BungarusJasciatus solutions of 0.02 M H3PO,t (anolyte) and 0.05 N NaOH venoms were purchased from Miami Serpentarium, Miami. (catholytc), with the anolyte in the upper chamber• After Florida. Separate milkings of the right and left fangs of electrolysis at 100 V for 10 min to discharge potentially a specimen of N,ju melanoleuca were a gift from Mr. Wilharmful persulfate, the samples were applied through the liam Haast, Miami Serpentarium. Naja melanoleuca venom anolyte onto the top of the gel as a high density buffered was also obtained from Jonathan Leakcy Ltd.. Baringo, solution (4",/0 ampholyte solution, pH 7-10). After sample Kenya, East Africa, for comparative studies. All venoms application, the voltage was increased and maintained at were stored at - 16 C. 2(X)V for I hr and then raised to 300V for 4 h r more. For disc gel electrophoresis, the reagent sources were: The staining procedure for protein involved immersing Tris (hydroxymcthyl) aminomethane and glycine, Sigma the gels in 12.5% trichloroacetic acid solution for 10 min Chemical Co.: cyanogum 41 gelling reagent, EC Apparatus . to precipitate proteins, followed by transferring the gels Corporation N,N.,\",N'-tetramethylethylenediamine, East- to a Coomassie brilliant blue G-250 colloid suspension, man Organic Chemicals. and ammonium persulfate, J. T. which was made by vigorous mixing of 1 ml 0.25% Coo193

194

St'-RAV LH:, JENEHIR Lit.: LATIA AND Wlt,t ARD B. ELt U)Tr

massie brilliant blue (; 250 aqueous solution with 20 ml 12.5°,, trichloroacetic acid. and allowed to stand for 1 2 hr. The gels were then transferred to a 5". acetic acid solution. allowing the d~c to penetrate into the gel matrix and bind to the proteins (Diczcl et al., 19721. Histochemical staining with BCNCI was carried out by incubation of the gel m a staining solution 120 mg BCNCI and 30 mg diazo blue B dissoh.ed m 50 ml l r i s CI buffer. pH 7.4, 0.(15 M) for 10 30 rain. fi~llowed by washing with 7". acetic acid Activity was indicated b~ purple bands resulting from the reaction of diazo blue B witb the released fl-naphthol (Uriel, 1961}, 1963: Wieme, 19651. Activity was assayed by a spectrophotometric method of Ellman et al. 119611 v,hich t.scs acetyltbiocboline as substrate. The reaction was started by: adding the enzyme solulion and the increase in absorbance of the reaction mixture ~.;ts recorded for 3 5 rain ~ith a Car.,, 14 spectrophotomcter. The cholinestcrg,se activit) ~.as expressed m units (u = mmoles of st, bstrate hydrol',zcd hr- i mg t proteinl and cMculated as I'ollo~ss: Unit - (/%A.mm) x (~) x II0 ~ El x (I .amount of enz)me in mgl where E (molar absorbtivikxl - 1.36 x It) a tool -1 cm t of the colored product and ~A is the difference m absorbance between the test sample and a suitable blank. RESL'L1 S

Disc +,}el eh,ctrophoresis of Naja melanoleuca. Bungarus fasciatus aml Naja naja naja ucetvh'holinesterase T h e acetylcholinesterase of Nak+ me/anoleuca v e n o m from Miami S e r p e n t a r i u m was present in two bands. I and If. in the o r d e r of decreasing positive charge as s h o w n in Fig. lB. The v e n o m of Naja melanoleuca obtained from K e n y a had a specilic acti,,'it', of only 40",> of that fronl M i a m i S e r p e n t a r i u m (Table 1). Both venoms had two similar acctylcholinestcrase activity bands after disc gel elcctrophoresis (Fig. I B and C}, with m o r e actix.ity in band I1 than in band I. Band II activity is present in a higher a m o u n t m the sample from K e n y a as s h o w n by scanning of the disc electrophoretic gels aflcr electrophoresis and cytochemical reaction with B ( ' N C I .

iiiiiiii

I

Table I. Comparison of specilic activities of \ . me/am*It+.'Zt('a

', cn onl

S[~citic acti~ it) tmnloles thiocholine released hr mg ,.h-) Vet) Venom Venom from Kenya I+rom Miami (4 mg.mll 14 mg roll

Volume of ~.enom added to the reaction medium l/d) 1

0.81

2 3 average

0.87 0.84 0.84

2.1 2.0 2.o 2.0

The v e n o m milkings from individual fangs of a

Naja melanoleuca at Miami S e r p e n t a r i u m also have the same two acetylcholinesterase activity bands (Figs. l l) and E) as present in the pooled venoms o b t a i n e d from K e n y a and Miani. Purified Bun qarus.ld.sciatt+x and /Vqja mOa m!ja acetylcholinesterases s h o w e d four activity b a n d s after disc gel electrophoresis {Figs. 2 and 3). The n u m b e r o f isozymes detectable by disc electrophoresis is the same in crude venom, partially puritied material and the isolated ACE.

Ion exchan.qe chromato+.#'aplo of Naja m c h m o l e u c a and Naja naja naja on DEAL Sephadex and SE.-25 Sephadex The possibility of charge isozymc-s' of A ( ' E was noted during puritication (Lee & Elliott. m preparation) of Naja melamdeucu acet 31cholinesterase b,, ion exchange c h r o m a t o g r a p h 3. on D E A E Sephadex in that the acetylcholinesterase isozvmcs tended to elute at different ionic strengths. Disc gel electrophoresis of these pooled fractions showed that the separation was good at t:'+o extremes of the activit5 peak as showt~ m Fig. 4, where the A C E ' s wcrc separated into l, I + II and 11. W h e n pooled fractions v+ith high A C E activity flom the c h r o m a t o g r a p h y of the v e n o m of ,\"aja nak~ m&l were eL, ted from a SE-25 Scphadcx c o l u m n with a Na('I gradient, the activity resolved into two peaks with A C E activity as s h o w n m Fig. 5.

~ ] ==,=,=,=== i

B

D

Fig. 1. The separation of crude N,ja melamdeuca venoms by disc gel electrophorcsis. Gels were run at 4 mA per tube for I hr. A. 200#g venom from Kenya. stained v, ith Coomassie brilliant btuc G 25(I. B. 2(X)#g venom from Miami. C 400 l
A

B

C

Fig. 2. Tile separation of acet)lcholmesterase isoz~mes of Bunqarus.lusciatus venom b,, disc gel electrophoresis. (icls were run at 4 mA per tube for 2 hr. ,\. 250 lig crude venom. B. 5.ug pure enzyme. Both A and B wcrc developed by the histochcmical reaction with B('N(.'I. ('. IOllg pure enzyme stained with ('oomitssie brilliant blue (i-25(1.

lsoenzymes of elapid acetylcholinestcrases

195

Effect of polyacrylamide gel concentration ration of ACE in eleetrophore.sis

on

the mi.q-

Variations in polyacrylamide concentrations from 6 10";, dramatically affected the "'sieving" properties of the gel. The plot of electrophoretic migration of the bands (I and II) of ACE activity present in Naja melanoleuea venom as a ffmction of acrylamide concentration appeared as two parallel lines (Fig. 61.

Gel permeation

A

B

chromatography

pattern qf Naja

melanoleuca t:enom

C

Fig. 3. The separation of partially purified and purified acetylcholinesterasc isozymes of Nqja mda by disc gel electrophorcsis. Gels wcrc run at 4 mA per tube for 2 hr. A. ItXI ttg partially purified enzyme developed by the histochcmical reaction v.ith BCNCI as was B. 10~ug purified enzyme. C. Same as B but stained with Coomassic brilliant bluc G 25(I.

Acetylcholinestcrasc isozymes of Naja mehmoleuca eluted as a symmetrical peak with gel permeation chromatography on Sephadex G - 1 5 0 (kcc & Elliott. in preparation). The relative concentrations of these isozymes in the individual fractions (e.g, fractions 60. 63, 67 and 71) were invariant throughout the peak as shown in Fig. 7, with disc gel patterns scanned after the histochcmical reaction.

E c O

0.4 M N o C I

t-<~

(A)(BI

._._>

(A)(B)

(A)(B)

tlJ ¢,.) z < GD oc 0

t

40

FRACTION

80

NUMBER

Fig. 4. Gradient clution of crude Naja melanoleuca (30Omg) venom from DEAE Sephadex cohnnn (I.7 × 30cm). The active peak can bc divided into isozyme 1, isoz)me I ~- II mixture and isoz)mc 11 fractions which wcrc fractions 59 (¢,, 65- 75 and 76-83. Disc gel clcctrophoresis of active pooled fractions, running conditions the same as in Fig. 1, are shown al'xwe the curve. A's were stained with Coomassie brilliant bluc G 250 and B's were reacted with BCNCI.

¢: c 0 cO t%1

0.15 M NoCI >

k<

(A)tB)

kxl 0 z <[

0 cO ,¢[

L

(A) (B)

(A) (e}

II

40

IO FRACTION

NUMBER

Fig. 5. Gradient elution of pooled fractions from venom of Naja naja nqia with high acetylcholinestcrase activity from SE 25 Sephadcx column. NaCI, 0.15 M gradient was added into the eluting buffer after the first material was completely eluted out of the column. The fractions in sections I, II and Ill were examined by disc gel clectrophorcsis. A's were stained with protein staining and B's were reacted with a substrate reaction of acetylcholinesterase.

196

SU-RAY Llil!. JI-NEFER LIU L A ] ' r A AND WII.LARD B. EI.tlOTT

140 0 0

E

n~

.J

'i

0 0

I00

i

i

6

GEL

8

I0

CONCENTRATION

Fig. 6. The effect of gel concentration on the mobility of acetylcholinesterase isozymes I and II from Naia melanoleuca venom.

lsoelectric focusinq ~acetylc/tolinesterase i~7pol)'~wry/amide ,qels Since with disc gel electrophoresis, the ACE bands were broader than one would expect for a singlc protein and appeared to have fine structure, isoelectric focusing was used. Figures 8A and B show the results obtained with the venoms from Bun,qarus Jhsciatus and Naja melanolet, ca. The venom of Naja melanoleuca had eight ACE activity bands (two groups of four each) with isoelectric points ranging from 4.2 to 5.2, while the venom of Bun#arus fiLsciatus had ten ACE bands with isoelectric points from 4.3 to 5.3. DISCUSSION

According to Markert (1968), "'Once the accepted criteria for defining a collection of molecules as an enzyme have been successfully applied, and if these molecules by any means (electrophoretic, chromatographic, solubility, immunochemical, etc.I can be separated into distinguishable types, then these types represent isozymic forms of the enzyme.'" The evi-

dence presented above (Figs. 1, 2 and 3) suggests the presence of two ACE isozymes in the venom of Na/a mehotoleuca and four in the purified Naja naja naja and Bungarusfttsciatus preparations demonstrable by disc gel electrophoresis. Naja melanoleuca venoms from different places have different spccific activities of ACE in the crude venoms (Tablc 1). This may be due to the extraction of venom by differcnt techniques or contamination of the venom by protein matcrials from mouth secretions (Minton, 1969; Gans & Elliott, 1968). Although commercial venoms may be excellent sources of enzymes for use in research, there is a growing body of information indicating that changes occur in some components almost immediately following the c.icction of the vcnom from the gland (Gans & Kochva, L965; Gans & Elliott, 1968). The venoms from individual fangs of the same Naja melanoleuca have the same two ACE activity bands in disc gel clcctrophoresis as are present in the whole venoms (Figs. I D and E). Thus. each of the venom glands in a single specimen of Naja melano/euca can secrete two ACE activity bands separable by disc gel electrophoresis. The ratio of activity in band I to that in band 11 is thc same for the venom from the right fang and left fang (Figs. 1D and E). There are significant differences among the pooled venom from Kenya and Miami, and the pooled venom and the single fang milkings (1-ig. 11. All of them havc more activity in band 11 than in band I. Band II activity is present in a higher amount in the scparate milkings of right and left fangs. This may be caused by milking in different seasons, places and,'or may be under genetic control. The cxistence of multiple forms of ACE in crude venoms of Naja ,telanoleuca, Naja naja nqja and Bun.qarus fasciatus (Figs. 1, 2 and 3) is the same as in purified enzyme (Lee & Elliott, in preparation). This implies that these isozyme species are not artifacts occurring in the process rcquired to isolate them. Ion cxchange chromatography of N~ja melanoleuca and Naja naja naju venoms on DEAE and SE--25 Sephadex partially separated these isozymes with large portions of overlap (Figs. 4 and 5). The isozymes of Naia melano/euca ACE were shown to be charge isozymes by analysis of electrophorctic migration of the bands of ACE activity in disc gel as a function of acrylamide concentration.

m m m FRACTION

67 .......

i~// ~l II

I^LII^~I .......

ON 6 0

A

Fig. 7. Scans of disc gel patterns of thc fractions 60, 63, 67 and 71 of the ACE peak from the Scphadex G 150 column.

B

Fig. 8. Isoelectric focusing of acetylcholinesterase isozymes. Venom samplc.s from Bunqaru,~fasciatu.s and Naja melanoleuca were submitted to isoeleclric focusing in A and B and developed for enzymatic activity.

Isoenzymes of elapid acetylcholinesterases According to Hedrick & Smith (1968), charge isomeric proteins give a parallel family of lines when the protein mobility relative to the dye front is plotted vs acrylamide gel concentrations. The plot in Fig. 6 indicates that ACE isozymes in Naja melanoleuca have the same molecular weight but different charges. Scanning of disc gel electrophoretograms of selected fractions eluting in the active peak from a Sephadex G 150 column (Fig. 7) reveals a constant ratio between the two groups of isozymes, indicating that all of the isozymes have essentially the same molecular weight. When the venoms of Naja melanoleuca and Bun.qarus fasciatus were subjected to isoelectric focusing, the venom of Naja melanoleuca resolved into eight ACE bands (two groups of four each) and the venom of Bungarus fasciatu.s, into ten. The ACE isozymes demonstrated here comprise the major aryl esterases of the venoms. Since this enzyme plays no role in the neurotoxic action of the venoms (Ghosh et al., 1941 : C h a n g & Lee, 1963) the function of this enzyme in venom remains obscure. The reason for the complexity of this enzyme in snake venoms may have its own physiological significance. REFERENCES

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