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Subunit composition of horse liver alcohol dehydrogenase
ARCHIVES
OF
Subunit
BIOCHEMISTRY
AND
BIOPHYSICS
131, 2882%
Composition
of Horse
REGINA
PIETRUSZKO
The Nobel
Medical Received
Institute, December
(1969)
Liver
Alcohol
Dehydrogenase
AND HUGO THEORELL
Department
of Biochemistry,
12, 1968,
accepted
January
Stockholm,
Sweden
5, 1969
Horse liver alcohol dehydrogenase, LADH, occurs in multiple, electrophoretically separable forms. The subunit composition of the three main forms is described in this paper. The enzyme LADHE (EE) consists of two identical halves (E, for activity with ethanol). The enzyme LADHss (SS) consists of two other identical halves (S, for activity also on steroids). The electrophoretically intermediate form LADHs (ES) is a hybrid consisting of one subunit E and one subunit S. Various conditions of dissociation and reconstitution of ES are investigated. Although in some of these conditions the relative amounts of the isoenzymes reconstituted are variable, formation of only three isoenzymes is observed. The results suggest that the quaternary structure of LADH is dimeric. The basis for the use of E and S in the nomenclature of the other isoenzymes of LADH is discussed.
In a previous paper (1) we have shown that subunit associations are responsible for the formation of some of the isoenzymes of LADH.’ One of theseisoenzymes (LADHs (2)) or isoenzyme 1 (3) is active with both the classical substrates of LADH and steroid substrates and consists of two kinds of subunits. We suggested that one kind of these subunits is associated with the ethanol activity of LADH (subunit E) and another kind with the steroid activity (subunit S). The study also showed that on dissociation and reconstitution of the enzyme consisting of E and S subunits a homogeneous enzyme composed of E subunits could be formed. It appeared probable, therefore, that an enzyme consisting of S subunits might be formed by the same procedure. The formation of such an enzyme is described in this paper. By the recombination of E and S subunits three LADH isoenzymes are formed of which two are
homogeneous (EE and SS) and a third one (ES) is a hybrid consisting of equal amounts of E and S subunits. In some horse liver extracts, enzymes more basic than the hybrid enzyme ES (2,3) and exhibiting greater activity towards steroids were already observed by A. Akeson and S. Taniguchi in this institute in 1966. The identity of one of these enzymes with the homogeneous enzyme SS obtained by dissociation and reconstitution of the hybrid enzyme, has now been established. As a result of these findings, three isoenzymes of LADH can be now represented in terms of the electrophoretically separable subunits E and S. A new nomenclature for isoenzymes of LADH based on these findings will be proposed.
1 The following abbreviations are used: horse liver alcohol dehydrogenase, LADH; nicotinamide adenine dinucleotide, NAD ; reduced nicotinamide adenine dinucleotide, NADH; nitroblue tetraaolium, NBT; phenazine methosulfate, PMS; 17P-hydroxy-5&androstan+one, 5 @DHT, ethylene diamine tetracetic acid, EDTA.
288
MATERIALS
AND
METHODS
Phosphate salts (Na2HP04 and NaHzPOa “nach Siirensen”), zinc chloride (pro analysi) and urea (pro analysi) were from Merck, Darmstadt, Germany. Urea was dissolved in hot water, mixed with MB3 double-bed ion-exchange resin and filtered, while hot, through a sintered glass filter. The first batch of crystals was collected by filt,ration and dried at 70” for 24 hr. Dry crystals were stored at room temperature in a stoppered bottle, and urea solutions were made fresh from these
HORSE
LIVER
ALCOHOL
crystals about 1 hr before expcrimellts. Both cyclohexanone aud acetaldehyde, from Kebo, Sweden, were redistilled and stored at 4” prior to use. Fresh sohltions of 152 acetaldehyde in 0.03 M phosphate buffer, pI1 7.0, were prepared before each experiment. 2.Mercaptoethanol (for chemical pllrposes) from Eastmall Kodak, USA, was used as supplied or redistilled under reduced pressure with nitrogen capillary, b.p. 54-55” at 40 mm Hg. NAD, NADII, NBT, P&IS, and Trizma base were from Sigma, USA. Ethanol, 95’;; rectified, was supplied by AB Vin- & Spritcentralen, Stockholm. Steroids: 5pDIIT aud 3p-OH-5p-androstan-17ketotle from Rlarm Research Laboratories, U. 8. A., alld Preparations Laboratory, Sew York U. S. A., respectively, were used without further purification. Enzymes: EE, ES, and SS were purified by ion-exchange chromatography on diethylaminoethgl cellulose and carboxy methyl cellrdose ~olun~~rs until they appeared as single components on zone electrophoresis. Enzymes EE alld ES were pure wth respect to other LADH isoenzymes and also free from foreign protein. The enzyme SS used in reconstitution experiments was free from other isoenxymes but it contained a considerable amount of foreign protein, with respect to which it was 34y0 pure. Enzyme SS used for subunit electrophoresis and estimation of specific act,ivity was electrophoretically pure. Condition,s for dissociation and reconstit~ction of the enzll,nes. The procedllre employed here was essentially the same as that used by Drum el al., 4. The enzymes at 200-500 rg/ml were exposed to 8 Y urea in 0.1 M phosphate buffer, pH 7.5, containing 0.1 M 2-mercaptoethanol (unless stated otherwise) for 8 min at room temperature (19-23”). At the end of 8 min the enzymes were diluted by t,he addition of 9 vol of 0.1 M phosphate blrffer, pH 7.5, containing 0.6 mM XADH, and G PM ZnClz . The dilllted enzymes were incubated at room temperature for 2 hr and then dialyzed for 18 hr against 0.05 M phosphate bluffer, pH 7.5, at 4”. Controls Tere exposed to 0.1 M phosphate buffer, pH 7.5, instead of urea, then diluted, incubated, and dial)-zed in the same way as the reconst,ituted enzymes. Spectrophotometric data on reconstituted enzymes and controls were obtained on dialyzed solutions using a Cary 14 spectrophotometer at 1: 10 scale expansion. Enzy?ne assays. Enzyme assays were done in l-cm cuvettes at 340 rnp at 23.5” in 3 ml total volume, in one direct,ion using NAD, 0.45 mM; ethanol, 8.6 mnf; in glycine buffer, 0.062 M, pII 10.0, and in the opposite direction using NADII, 0.175 mnr; 5flDIIT, 0.11 mM; cyclohexanone, 0.11 mM; acetaldehyde, 1.2 mu, ill 0.03 M phosphate bluffer, pH 7.0.
DEHYDROGEXASE
259
E1ectrophore.si.s. The dilute, reconstituted enzymes and controls were concentrated by vacuum dialysis against 0.05 M phosphate bul‘fcr, pII i.5, to volumes rangitrg from 0.05 ml to 0.1 ml and adjusted by dilution to the same or similar activity/ml. Starch gels were made by adding 11.25 g starch (Connaught, Canada) per 100 ml of 0.025 M Tris HCl buffer, pH 8.5. Electrophoresis was done on horizontal starch gels at 4” for 15-20 hr at 200 V constalrt voltage. The gel dimensions were: 25.5 X 8.6 X 0.5 cm. In the electrode vessels 0.3 RI Tris HCl bufl’er, pH 8.5, was used. Cellulose acetate electrophoresis was carried out at roan, t,emperature in a Beckman apparatus at 0.175 mA constant, current per l-cm width of ~llulose acetate st,rip, for 2W.O min. Cellulose acetate was soaked in 0.025 M Tris IICl buffer, ~118.5, while the electrode butier was 0.05 1~. After completion of electrophoresis the following solution was Ilsed for activity staining of gels a,nd cellulose acetate strips: L)P?: 18 mg; NBT 10 mg; PMS 1 mg, ethanol ‘35:6 0.05 or 0.1 ml; So-OI-I-5p-IT-ketoandrost,an 2 mg per 25 ml of 0.05 M Tris HCl buffer, pH 8.5. When st,eroid was used alone as substrate, it was dissolved in 0.1 ml of acetone, when used together with ethanol, it was dissolved iI1 0.1 ml of ethanol. Starch gels, after slicing, were incubated in the above solutions at room temperatllre, in the dark, for varying lengths of time, depending on the reclllirement of the experiment and the initial load of t,he enzyme (15 min to 4 hr). Cellulose acetate strips were developed at room tempcrature, in the dark, by overlaying with another cellldose acetate strip soaked in the activit,y stain. For protein staining, nigrosin (obtained from (;. T. Gurr, London, England), 0.02cG w/v solution in 407, v/v ethanol in water containing 2.5’:; w/v trichloracetic acid, was r~scd for starch gels, and 0.002~~ w/v solution in 2(., v,:v acetic acid in water was used for cellulose acetate strips. Starch gels or cellulose acetate strips were placed in t,heir respective staining solutions and then left in closed boxes overnight. Excess stain was washed Cellldose away with 5X v/v acetic acid in water. acet)ate strips were also stained with Ponceau S, using stain and instrclctions supplied by Beckman, u. 8. A. Determination of the composition of isoenzymes after electrophoreais on cellulose acetate. Proteitl bands on cellulose acetate, visible after staining with ponceau S, were cut out and the adsorbed stain was eluted into l-ml vol of 0.1 N NaOII contaiued in IO-ml centrifrlge tribes. After the elution was complete, the bllle solutions were ceutrifuged t,o remove some celllllose acetate fibers. The amolmt of stain corresponding to variolls protein bands was determined colorimet-
a From b Values
(3 ,---_* L--.2
horse
Present in
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
commerf’a’ preparations
+
liver
Present in
SEPARATION
LADHs
LADHE
2
+
2
and ion-exchange of enzyme activity
+
f
+
f
+
2A
1
+
+
4 3
-I-
5
Ethanol activity
per
ss
ES
EE
in 3-ml
Nomenclature relating to subunit composition
-
ES’
ES”
EE’
EE”
composition
TsLW&i;
ISOIZNZYMES
per
mg enzyme
min
volume
SS’ 6,700h
130,000
250,000
Acetaldehyde
substrate
SEPARATED
SS” 4,900b
2,300
10-300
SODHT
with
OF THE
? 4,ooob
7-10,000
15 ) 500”
Ethanol
activity
PROPERTIES
Specific
SOME
?
?
I AND
chromatography3. = change of 0.001 OD
+
+
f
-
-
-
Steroid activity
TABLE ELECTROPHORESIS
y;ry;5cc!lan‘ Ref. 3
ON ZONE
Nomenclature as in Ref.
PATTERN
Identity confirmed by re-electrophoresis Identity not yet confirmed. One unit described in Materials and Methods. Ref. 2. from a single estimation.
<----. ..___, i---.,-,2
0
on zone electrophoresis
Isoenzymes of LADH
LADH
J
I
I
I
J }
in conditions
ss
ES
EE
Group subdivision
F
ii
8
G
$ u
t!
Ti K
cd E c3
HORSE LIVER ALCOHOL DEHYDROGENASE rically using at 540 mr.
a Beckman
1)U
RESULTS XOMENCLATURE OF LADH
spectrophotometer
ISOEN~YMES
LADH occurs in multiple isoenzymic forms (l-3). Some horse livers contain at least nine electrophoretically separable components (5) which migrate toward the negative electrode on starch gels at pH 5.5. A schematic representation of the electrophoretic separation pattern of LADH on solid supporting media is given in Table I. Since independent investigations on some of the LADH isoenzymes were carried out concurrently by two groups (1, 3) two different nomenclatures were used. They are presented in the table together with some properties of these isoenzymes and the proposed new nomenclature. In our previous paper (1) we showed that one of the isoenzymes consisted of two elec-
FIG. 1. Inactivation of ES in 8 M urea containing 0.1 M 2-mercaptoethanol in 0.1 M phosphate buffer, pH 7.5. The enzyme activity was assayed with 6.9 IIIM cyclohexanone. Other components of the assay system were as described in text. The control rate in 0.03 M phosphate buffer containing 0.133 M urea and 0.0016 M 2.mercaptoethanol was 23$& of that in the phosphate buffer alone, which indicates that urea and mercaptoethanol even in these low concentrations inhibit the reaction velocity.
291
trophoretically separable species of subunits. As a result of previous and present investigations we know that the more negatively charged subunit species (subunit E) is common to t,he hybrid isoenzyme (isoenzyme 1 or LADHs) and the homogeneous isoenzyme (isoenzyme 3 or L~DHE). The more positively charged subunit species (subunit S) is common to the hybrid isoenzyme and a homogeneous isoenzyme which exhibits even higher steroid activity than the hybrid. In the new nomenclature the hybrid isoenzyme, consisting of two differcnt subunits, is named ES, t,he homogeneous more negatively cha,rged isoenzyme, EE, and the homogeneous and more positively charged isoenzyme, SS. The use of E and S in the nomenclature for the other isoenzymes of LADH will be discussed. DISSOCIATION AND RECONGTITUTION OP EE, ES, AND 8s hxtivation of ES ia urea. ES (0.5 mg/ml) was dissolved in 8 M urea, 0.1 M phosphate buffer, pH 7.5, and 0.1 ~2-mercaptoethanol. Samples of 0.05 ml mere taken at different time intervals, and the enzyme activity was measured with cyclohexanone as substrate. The assay system contained 0.133 M urea and 0.00166 M 2-mercaptoethanol, in addition to the usual components described in Materials and Methods. The time course of the inactivation of ES is shown in Fig. 1. The results are expressed as percentage of the activity of the undissociated control. At times longer than S min the activity remained at a constant level of about 10% of the control, thus suggesting that t’he dissociation of ES may not be going to completion. Since in our experiments it was necessary that ES should be fully dissociated, 1y-e developed in collaboration wit’h Dr. J. Iioepke (6) an electrophoretic procedure by which dissociated and denatured J,ADH could be separated rapidly from the native undissociated enzyme. Using this technique we found that after 7 min exposure to S M urea containing 0.1 nr 2-mercaptoethanol ES, EE, and SS were no longer present in the solution. It is assumed, therefore, that the activity exhibited
292
PIETRUSZKO
AND
THEORELL
phoretic pattern of SS after dissociation and reconstitution. It is identical with the control SS in Fig. 2A. A similar comparison is made in Fig. 3B and 3A after EE was subject’ed to the same procedure. Fomnatiow of EE: and SS from ES. When pure ES was dissociated and reconstituted three enzymes were formed. In addition to ES, EE, and SS appeared. This is shown in Fig. 4, where the more negatively charged enzyme formed from ES is identified elec-
FIG. 2. Dissociation and reconstitlltion of SS. Starch gel after electrophoresis was stained for enzyme activity using a mixture of 5@DHT and ethanol as substrate. A, SS control, B, SS after dissociation and reconstit,rltion, C, ES after dissociation and reconstitution.
by the ES at times longer than 7 min is due to the partial reactivation of the enzyme during the assay. According to Hamburg’s (7) optical rotatory dispersion measurements on LADH (obtained from C. F. Boehringer & Soehne GmbH, Germany,) even shorter time (4 min) and less concentrated (7 M) urea were sufficient for complete dissociation. Dissociation and reconstitution of AS’Sand of EE. The results demonstrated in Figs. 2 and 3 show that SS and EE when subjected separately to t’he procedure of dissociation and reconstitution reappear with unchanged properties. Figure 2B shows the electro-
A
B
C
FIG. 3. l>issociation and reconstitution of EE. I, gel stained for enzyme activity using a mixture of @I)HT and ethanol as substrate, II, gel stained for protein with nigrosin. A, EE control, B, EE after dissociation and reconstitution, C, 50-8076 saturated ammonium sulfate fraction of horse liver extract, used as marker showing all isoenzymes of LAl)H except SS’ and SS”.
C
A
FIG. 4. Dissociation and reconstitution of ES. I, gel stained for enzyme activity using a mixture of 5pDHT and ethanol as substrate; II, gel stained for protein with nigrosin. A, ES control; B, ES after dissociat,ion and reconstitution; C, EE control.
HORSE
LI\-ER
ALCOHOL
DEHYDROGENASE
293
1: 1. The more negatively charged subunit of ES occurs exclusively in EE (Fig. GC), while SS consists exclusively of the more positively charged subunit (Fig. GA). DISSOCI~TIOK
OF
EE
ASD
I’KESENCE ASD ABSEXCE 2-l IEKC.\PT~ETH~~K~L
c FIN:. 5. Dissociation and reconstitkltioll of a misttIre of EJZ and SS Cellulose acetate after elect rophoresis was stained for enzyme artivit? with ethanol as substrat,e. A, mixture of BE and SS, colltrol; 13 and C. JXE and 88 after dissociation :tlld recollstiflltion; C, ES control.
t’rophoreticall;with corkol EE. The electrophoret,ic identification of the more positively charged enzyme, formed from ES, shown in Fig. 2. (Compare 2A and 2C). Forrti,ation of ES jr017~ a mixture of EE at/t/ Shy. In Fig. 5 (5B and 5C) the formation of Id% is demonstrated after subjection of a mixture of EE and SS (Fig. 3) to the dissociation and reconstitution procedure. ES, formed from the mixture of EE and SS is identified electrophoretically with the control ES (compare Figs. 5B and 5C with Fig. 51)). hkl~~opl~o~~esis of EE, ES, and SX in the dissociated state. EE, ES, and SS were exposed separately to X M urea in 0.1 M phosphate buffer, pH 7.5, containing 0.1 M 2-mcrcapt,oethanol and 0.01 M EDTA at room t~emperature for 1 hr. EDTA was used here to remove ZI?+ from the subunits (4) in order to avoid possible interference of bound Zn’f with the subunit electrophoresis. The electrophoretic patterns of dissociated El;,, ES, and SS arc shown in Fig. 6. Fig. GB shows the separation of two different, subunit.s of ES. The relative percentage composition of ES was determined colorimetrically after protein staining. In t,\vo experiments, \ve found 52 and 50% subunit’ E and 4S and 5OC; subunit S. ES consists, therefore, of E and S subunits in the ratio
ES
IX
THE
OF
Drum et al. (4) believed, on the basis of ultracentrifugation experiments, that LADH is dissociated into subunits of 40,000 RIW (“dime? ~20,~ = 2.4) or into subunits of 20,000 MW (“monomer” ~20,~ = 1.5) depending on the dissociation conditions. Electrophoresis of subunits oj EE and ES it2 “mo72omer” at/d “tlin7.er” cotditions. EE and ES, 10 mg/ml, were exposed to S M urea in 0.1 x phosphate buffer, pH 7.5, (“dime? conditions) (4) or in the additional presence of 0.1 M 2-mercaptoethanol and 0.01 M EDTA (“monomer” conditions with total Zn2+ removed) (4) at room temperature for at least 1 hr before electrophoresis. Dissociated enzymes were electrophoresed in S Y urea for “dime? conditions and in S I\/ urea containing 0.1 M 2-mercaptoethnnol for L‘n~onomer” conditions. Electrophoresis was carried out at room t’emperature at a constant current of 0.175 mA per cm width of cellulose acet’ate strip for 30 min in various buffer systems. The results of electrophoresis of dissociated EE and ES in the “monomer” and ‘Ldimer” conditions are shown in Table II. In both conditions EE migrates as one
+
-
ABC FIG. 6. Electrophoresis of E:E, ES, arld SS iu the dissociated form. Aft.er dissociation, electrophorzsis in 8 M tIrea containing 2.mercaptoethanol in glycille brlffer, pH 9.6 (for details see Table II), on celllllose acetate srlpporting mediums. A, SS; B. ES; C, EE stained with nirtusin.
294
PIETRUSZKO
AND TABLE
ELECTROPHORETIC
OF LADH
SEPARATION
s~~;i~;$~d cont. buffer (Ml
Separation
of LADH
subunits
Buffer added to 8 M urea and 0.1 M 2-mercaptoethanol Glycine NaOH Glycine NaOH Tris-HCl Sodium phosphate Sodium phosphate Sodium phosphate Sodium acetate Sodium acetate Separation
of LADH
subunits
Buffer added to 8 M urea alone Glycine NaOH Glycine NaOH Sodium phosphate Sodium phosphate Sodium phosphate
in “monomer”
0.025 0.025 0.025 0.0125 0.0125 0.0125 0.025 0.025
0.025 0.025 0.0125 0.0125 0.0125
@Mixture pH was measured on mercaptoethanol. b Migrate toward positive electrode,
IN
“MONOMER”
Sample applied near electrode + or -
Mixture PH’
AND
“DIMER”
Subunits distance migration (mm) E
CONDITWNS
Separation distance (mm)
S
Enzyrpes dissoclated (number of bands) EE
ES
conditions
0.05 0.05 0.05 0.025 0.025 0.025 0.05 0.05
in “dime?
II
SUBUNITS
.BuEer (M) In vessels electrode
THEORELL
-
10.5 9.5 8.9 7.8 7.8 7.0 5.8 4.8
11* 2b 4.5 5.0 2.5b 5.5 10 20.5
+ + + +
10” 1.5 7.5 6.5 1.25b 7.0 10 20.5
1.0 3.5 3.0 1.5 1.25 1.5 0 0
1 1 1 1 1 1 1 1
2 2 2 2 2 2 1 1
0 4.5 4.5 8.0 10.0
2.5 3.0 1.0 1.25 1.25
1 1 1 1 1
2 2 2 2 2
urea,
and/or
conditions
0.05 0.05 0.025 0.025 0.025 pH
-
10.17 9.57 8.5 7.67 6.6 meter
all others
after
+ + + mixing
not marked
band and ES as two bands, except for lower pH values where E and S subunits are not separable. Since in the “dime? conditions Zn2+ was not to be removed from the subunits (according to Drum et al. (4) removal of Zn2+ results in a further dissociation of the “dime?‘), the electrophoresis had to be confined to the pH values at which no loss of Zn2+ from the LADH molecule was likely to occur. Although the rate of electrophoretic migration of the subunits obtained in “dime? and “monomer” conditions is slightly different, the degree of separation obtained in buffers of comparable pH values is almost identical. The small variations in the absolute rates of migration are very probably due to technical imperfections. Since the “monomeric” subunits had their Zn2+ completely removed by EDTA during dissociation (4), while the “dimeric” ones
2.5b 1.5 3.5 6.5 8.5
-
buffers
migrate
toward
with
8 M negative
2-
electrode.
had all their Zn2+ intact (4), we attempted a separation of the EE “monomers” and “dimers” by simultaneous electrophoresis. Thus, EE was dissociated in two batches: one in “monomer” and another in “dime?’ conditions. Both were then electrophoresed in several buffers containing 8 M urea. No difference in the rate of migration of these subunits was observed. An attempt was also made to separate “monomers” and “dimers” of EE by chromatography on Sephadex G-150 and G-200 columns but the void volumes with “monomers” were the same as those with “dimers” (6). Reconstitution of ES after dissociation in “dime? conditions. After dissociation in the absence of 2-mercaptoethanol, ES was reconstituted by dilution with phosphate buffer containing NADH (0.6 mM) and 2-mercaptoethanol, (0.01 M) (the latter was
HORSE TABLE EFFECT THE
0 8 15 :
ALCOHOL
III
OF DISSOCIATION RECONSTITUTION Substrates
Dissociation time (minj
LIVER
TIME
ON
OF ES (% reconstitution)
Acetaldehyde
Cyclohexanone
Ethano,
100 30.5 26.8 22.8 25.3 22.2
100 27.7 24 24 23.2 20.2
100 25.5 24.7 23 23 17.3
59 D HT
100 38 32.4 35.4 31.8
+
5432112345 Fro. 7. Effect of dissociation time on the isoenzyme composition resulting from combination of E :uld S subunits. I, starch gel stained for erlzymr artivity with a mixture of 5pDHT and ethanol :M substrate; II, stained for protein with nigrosin. 1, control before dissociation; 2, after 8 mill dissociation; 3, 15 min; 4, 30 min; 5,45 min.
used to protect the reconstituted enzymes against SH oxidation). After incubation, dialysis, concentration, and electrophoresis, formation of EE, ES, and SS was demonskated by protein and activity staining. The result was indistinguishable from that in “monomer” conditions (see Fig. 2). Efect of’ dissociation time on 7.econstitution o.f E’S. ES was subjected to the dissociating procedure in “monomer” conditions and different batches were diluted after different t,imes with a solution containing phosphate, NADH and ZnClz. After incubation followed by dialysis, the percentage of regain of the enzyme activity was determined spectrophotometrically, and Dhe isoenzyme composition was analyzed by electrophoresis.
DEHTDROGENASE
295
The effect of dissociation time on the regain of ethanol-, cyclohexanone-, acetaldehydeand 5@DHT-activity is shown in Table III and the effect of the dissociation time on t,he composition of isoenzymes in Fig. 7. The percentage regain of various activities of LADH isoenzymes is only slightly dependent on the dissociation times between S-60 min, and the composition of isoenzymes (Fig. 7) appears to be independent of the time of dissociation. E,J’ect of temperature on the reconstitution of ES. ES was dissociated at room temperature in “monomer” conditions. One sample m-as reconstituted by dilut,ion at 23” and another at 3S”. The reactivation, as measured by activity with cyclohexanone, was found to be 4O’X at 23” and 35 % at 3s” and in both cases only EE, ES, and SS were formed. E$ect of Zn2+ concentration of the reconstitution of E;S. ES (0.5 mg/ml) was dissociated conditions and then reconin ‘5nonomer” stituted by dilution with phosphate buffer, containing NADH and varying amounts of ZnClz. The effect of Zn2+ on the reconstitution of ES is shown in Fig. 8 and Table IV. Electrophoretic analysis at’ different concentrations of added Zn*+ (Fig. 8) shows that the increase of Zn2+ depresses t’he re-
FIG. 8. Ilccotlstittttion of ES at differetlt concentrations of Zn*+. Starch gel after electrophoresis was stained for enzyme activity using a mixture of 5pDHT and ethanol. Iteconstitkttion at different concentrations of Zn2+. 1 no Zn”+ added; 2. (i PM; 3, 18 ,.IM; -1-, 54 PM; 5, l& PM. The tmdissociated u)ntrol was the same as ill Fig. 7.
296
PIETRUSZKO TABLE
AND
IV
EFFECT ON ZN~+ ON THE RECONSTITUTION OF ES Percentage Percentage Ratio of ZnClz added reconstitution reconstitution SPDHT to during reconstitu- basedon cyclohexation (MM) cyclohexanone %A? none activity activity activity 0.0 6.0 18.0 54.0 162.0
Cont,rol
38.7 30.8 16.6 8.9 8.6 100
51.3 52.7 39.0 14.5 13.7 100
5.5 7.2 9.8 G.8 G.G 4.2
constitution of EE more than that of SS. The ratio of the activities with 5PDHT resp. cyclohexanone shows a maximum at 18 &!l Zn2+. The greatest regain of enzyme activity is obtained when no Zn2+ is added. In our experimental conditions 6 JVl Zn2+ inhibits the regain of the cyclohexanone activity and affects the composition of the isoenzymes formed from E and S subunits. Thus, with 6 PM Zn2+, EE: ES :SS was 1: 7 :2 while with no Zn2+ added, it was 1: 2: 1, as expected for simple statistical combination of two different subunits. Dissociation of ES in the presence of d-mercaptoethanol and EDTA. ES was dissociated in “monomer” conditions in the presence of 0.01 M EDTA (4). Under these conditions the E and S subunits lost their ability to recombine and neither ethanol nor steroid activity appeared on dilution. Electrophoresis showed that all the enzyme was present in solution in the denatured form and that no reassociation had occurred. This is in agreement with the results obtained by Drum et al. (4) on Boehringer LADH (mainly EE). DISCUSSION
In 1951 Theorell and Bonnichsen (8) established that each LADH molecule has two coenzyme-binding sites. Kinetic work carried out later in this laboratory strongly suggested that these two sites are functionally separate, equivalent, and independent (9). These studies were done on LADH which consisted predominently of EE. The evidence for two coenzyme-binding sites on ES (LADHs) was presented by
THEORELL
Theorell et al. (2) in 1966. Simultaneous investigations carried out in two laboratories (2, 3) demonstrated that the steroid activity of crystalline LADH was confined to ES and that EE, the major component, was almost devoid of this activity. Kinetic experiments with 3~OH cholanic acid used as a competitive inhibitor (a), or using mixtures of two substrates and by alternate product inhibition (3) showed that the reaction of steroids and classical substrates of LADH occurred at different catalytic sites. Work with antibodies (10, 11) estabblished that the two catalytic sites were located on t’he same protein molecule (ES) and indicated close structural relat’ionship between EE and ES. Further work (1) showed that EE and ES had a subunit in common (subunit E) and also that ES contained another subunit (subunit S) which was absent in EE. Present studies show that one of the naturally occurring isoenzymes of LADH (5) consists exclusively of these S subunits. Three isoenzymes of LADH (EE, ES, and SS) are formed by the hybridization of E and S subunits. ES is a hybrid consisting of equal amounts of these subunits. EE consists only of subunit#s E and SS of subunits S. Fluorimetric data of Theorell et al. (1968) (12) show that in the SS molecule there are also two catalytic sites which are equivalent with one another but different from those in EE. In our previous paper (1) we suggested that the steroid activity of LADH was associated with subunit S and ethanol activity with subunit E. This conclusion was derived from the study of the then available ES. Since small activity with nonsteroid substrates of the S site of this enzyme was obscured by the very high activity of its E site, it was thought t’hat the S site of ES was only active with steroids. However, later studies, especially after isolation of SS, have shown that although significant activity toward steroids is exhibited only by the isoenzymes containing subunit S, all LADH isoenzymes (including SS) are active with ethanol. The preliminary data on specific activities of EE, ES, and SS, obtained with 5PDHT, acetaldehyde, and ethanol, presented in Table I alongside with subunit composition, demonstrate
HORSE
LIVER
ALCOHOL
structure-activity relationship of these three isoenzymes. EE has high activity with classical LADH substrates, and only negligible, if any, activity with steroids. The activity of EE with ethanol and acetaldehyde is about twice that of ES. Although still active with ethanol, SS has a steroid activity which is twice tha,t of ES and very low activity with acetaldehyde. The activity of SS with acetaldehyde was in fact so low that it could have been caused by a small amount of EE or ES impurity. Extended kinetic experiments are required in order to clarify this interesting difference. The catalytic properties of ES are intermediate between those of EE and SS. This is in complete agreement with the subunit composition, since EE consists exclusively of subunits E, and SS of subunits S, while ES is a 1: 1 hybrid of these subunits. Since only SS and ES exhibit significant activity with the steroid substrates, the catalytic site concerned with the interconversion of steroids must be located on subunit S. The catalytic site concerned with the classical LADH substrates, but not with steroids, is present in subunit E. Chemical data on LADH, such as peptide mapping (13, 14)) end group determination (7, 15), X-ray data (16), and the fact that only two NADH molecules are bound per molecule of the enzyme (8), indicate that LADH is composed of two subunits. Further dissociation of LADH as reported by Drum et al. (4) cannot be reconciled with our evidence. After hybridization of E and S subunits, after dissociation of ES in Drum et al. (4) “monomer” and “dimer” conditions, we find only three LADH isoenzymes: homogeneous EE, hybrid ES, and homogeneous SS. If the quaternary structure of an enzyme is tetrameric and the reconstitution is random, five isoenzymes are expected from combination of two kinds of nonidentical subunits (17), if it is dimeric, only three enzymes can be obtained. Our attempts to show different subunits (6) in “monomer” and “dime? conditions (4) were unsuccessful (see above). Moreover, it is known that EE contains 28 cysteine residues per molecule (lS), 8 tyrosine residues, and 4 tryptophane residues (2).
297
DEHYDROGENASE
Recently Jornvall and Harris (1968) (19) in a study of tryptic digests isolated 14 unique cysteine peptides, 4 unique tyrosine peptides, and 2 unique tryptophane peptides per molecule of EE. This is expected for two identical 40,000 MW “dimers” but incompatible with a structure of four identical 20,000 MW “monomers.” In the recent Alfred Benzon Symposium in Copenhagen (10/g/68) (20) Dr. Howard Schachman showed new ultracentrifugation data on LADH demonstrating that the sedimentation constants under ‘Lmonomer” and “dime? conditions were the same, with values intermediate between those of Drum et al. (4) ~20,~ = 1.5 and 2.4. Prom the above considerations it appears that E and S subunits in both “monomer” and “dime? conditions represent halves of the LADH molecule. The combination of E and S subunits accounts for the structure of only three isoenzymes of LADH of the nine (5) present in horse liver (Table I). Since attempts to form isoenzymes other than EE, ES, and SS by the combination of E and S subunits have been unsuccessful, it appears that other subunit species must be involved in order to explain the composition of the remaining LADH isoenzymes. LADH separates on electrophoresis in a repeated pattern of three isoenzymes in three groups (5). EE, ES, and SS are the most positively charged in each group (Table I) and occur as the major components. As the structure of isoenzymes other than EE, ES, and SS is at present unknown, but the preliminary activity data suggest greater similarity between those in a given group than between individual members of separate groups, we have named these isoenzymes by attributing to them a structure similar to that of the known isoenzyme in the group. Thus, the isoenzyme migrating on electrophoresis just slower than isoenzyme EE is called EE’ and the one even slower EE”. Other isoenzymes of LADH have been named in an analogous way. REFERENCES IL, RINGOLD, H. J., LI, T-H., 13. L., .&U;SON, A., AND THEORELL, Nature 221,440 (I%%).
1. PIICTRUSZICO, VALLXE,
H.,
298
PIETRUSZKO
2. THEORF,LL, H., TANIGUCHI, S., AKESON, A,. AND SKURSKP, L., Biochem. Biophys. Res. Commun. 24, 603 (1966). 3. PIETRUSZKO, R., CLARK, A. F., GRAVES, J. M. H., AND RINGOLD, H. J., Biochem. Biophys. Res. Commun. 23, 526 (1966). 4. DRUM, D. E., HARRISON, J. H., LI, T-K., BETHUYE, J. L., AND VALLEE, B. L., Proc. Natl. Acad. Sci., U. S. 67,1434 (1967). 5. THEORELL et al. Unpublished data. 6. KOEPKE, J., WKESON, A., AND PIETRUSZKO, R., (1969) to be published. 7. HAMBURG, R. D., The Physical Properties of Horse Liver Alcohol Dehydrogenase. (Diss.) University of California, Berkeley (1966). 8. THEORELL, H., AND BONNICHSEN, R. K., Acta Chem. Stand. 6, 1105 (1951). 9. THEORELL, H., Harvey Lectures Ser. 61, 17, (1967). 10. PIETRUSZI~O, R., AND RINGOLD, H. J., Biochem. Biophys. Res. Commun. 33,497 (1968).
AND
THEORELL
11. PIETRUSZKO, R., RINGOLD, H. J., KAPLAN, N. O., AND EVERSE, J., Biochem. Biophys. Res. Commun. 33, 503 (1968). 12. THEORELL, H., AKESON, A., LISZKA, B. M., AND DF, ZALENSKI, C. W. T., (1969). Manuscript in preparation. 13. HARRIS, J. I., Nature 203,30 (1964). 14. LI, T-K., AND VALLE, B. L., Biochemistry 3, 869 (1964). 15. JBRNVALL, H., Acta Chem. Stand. 21, 1805 (1967). 16. BRAND&N, C. I., Arch. Biochem. Biophys. 112, 215 (1965). 17. MARKGRT, C. L., Science 140,1329 (1963). 18. YONETANI, T., AND THEORELL, H., Arch. Biochem. Biophys. 99, 433 (1962). 19. JBRNVALL, H., AND HARRIS, J. I., Abstract 769 FEBS Fifth Meeting, Praha (1968). 20. SCHACHMAN, H., Alfred Benson Symposium, Copenhagen (1968).
OF
Subunit
BIOCHEMISTRY
AND
BIOPHYSICS
131, 2882%
Composition
of Horse
REGINA
PIETRUSZKO
The Nobel
Medical Received
Institute, December
(1969)
Liver
Alcohol
Dehydrogenase
AND HUGO THEORELL
Department
of Biochemistry,
12, 1968,
accepted
January
Stockholm,
Sweden
5, 1969
Horse liver alcohol dehydrogenase, LADH, occurs in multiple, electrophoretically separable forms. The subunit composition of the three main forms is described in this paper. The enzyme LADHE (EE) consists of two identical halves (E, for activity with ethanol). The enzyme LADHss (SS) consists of two other identical halves (S, for activity also on steroids). The electrophoretically intermediate form LADHs (ES) is a hybrid consisting of one subunit E and one subunit S. Various conditions of dissociation and reconstitution of ES are investigated. Although in some of these conditions the relative amounts of the isoenzymes reconstituted are variable, formation of only three isoenzymes is observed. The results suggest that the quaternary structure of LADH is dimeric. The basis for the use of E and S in the nomenclature of the other isoenzymes of LADH is discussed.
In a previous paper (1) we have shown that subunit associations are responsible for the formation of some of the isoenzymes of LADH.’ One of theseisoenzymes (LADHs (2)) or isoenzyme 1 (3) is active with both the classical substrates of LADH and steroid substrates and consists of two kinds of subunits. We suggested that one kind of these subunits is associated with the ethanol activity of LADH (subunit E) and another kind with the steroid activity (subunit S). The study also showed that on dissociation and reconstitution of the enzyme consisting of E and S subunits a homogeneous enzyme composed of E subunits could be formed. It appeared probable, therefore, that an enzyme consisting of S subunits might be formed by the same procedure. The formation of such an enzyme is described in this paper. By the recombination of E and S subunits three LADH isoenzymes are formed of which two are
homogeneous (EE and SS) and a third one (ES) is a hybrid consisting of equal amounts of E and S subunits. In some horse liver extracts, enzymes more basic than the hybrid enzyme ES (2,3) and exhibiting greater activity towards steroids were already observed by A. Akeson and S. Taniguchi in this institute in 1966. The identity of one of these enzymes with the homogeneous enzyme SS obtained by dissociation and reconstitution of the hybrid enzyme, has now been established. As a result of these findings, three isoenzymes of LADH can be now represented in terms of the electrophoretically separable subunits E and S. A new nomenclature for isoenzymes of LADH based on these findings will be proposed.
1 The following abbreviations are used: horse liver alcohol dehydrogenase, LADH; nicotinamide adenine dinucleotide, NAD ; reduced nicotinamide adenine dinucleotide, NADH; nitroblue tetraaolium, NBT; phenazine methosulfate, PMS; 17P-hydroxy-5&androstan+one, 5 @DHT, ethylene diamine tetracetic acid, EDTA.
288
MATERIALS
AND
METHODS
Phosphate salts (Na2HP04 and NaHzPOa “nach Siirensen”), zinc chloride (pro analysi) and urea (pro analysi) were from Merck, Darmstadt, Germany. Urea was dissolved in hot water, mixed with MB3 double-bed ion-exchange resin and filtered, while hot, through a sintered glass filter. The first batch of crystals was collected by filt,ration and dried at 70” for 24 hr. Dry crystals were stored at room temperature in a stoppered bottle, and urea solutions were made fresh from these
HORSE
LIVER
ALCOHOL
crystals about 1 hr before expcrimellts. Both cyclohexanone aud acetaldehyde, from Kebo, Sweden, were redistilled and stored at 4” prior to use. Fresh sohltions of 152 acetaldehyde in 0.03 M phosphate buffer, pI1 7.0, were prepared before each experiment. 2.Mercaptoethanol (for chemical pllrposes) from Eastmall Kodak, USA, was used as supplied or redistilled under reduced pressure with nitrogen capillary, b.p. 54-55” at 40 mm Hg. NAD, NADII, NBT, P&IS, and Trizma base were from Sigma, USA. Ethanol, 95’;; rectified, was supplied by AB Vin- & Spritcentralen, Stockholm. Steroids: 5pDIIT aud 3p-OH-5p-androstan-17ketotle from Rlarm Research Laboratories, U. 8. A., alld Preparations Laboratory, Sew York U. S. A., respectively, were used without further purification. Enzymes: EE, ES, and SS were purified by ion-exchange chromatography on diethylaminoethgl cellulose and carboxy methyl cellrdose ~olun~~rs until they appeared as single components on zone electrophoresis. Enzymes EE alld ES were pure wth respect to other LADH isoenzymes and also free from foreign protein. The enzyme SS used in reconstitution experiments was free from other isoenxymes but it contained a considerable amount of foreign protein, with respect to which it was 34y0 pure. Enzyme SS used for subunit electrophoresis and estimation of specific act,ivity was electrophoretically pure. Condition,s for dissociation and reconstit~ction of the enzll,nes. The procedllre employed here was essentially the same as that used by Drum el al., 4. The enzymes at 200-500 rg/ml were exposed to 8 Y urea in 0.1 M phosphate buffer, pH 7.5, containing 0.1 M 2-mercaptoethanol (unless stated otherwise) for 8 min at room temperature (19-23”). At the end of 8 min the enzymes were diluted by t,he addition of 9 vol of 0.1 M phosphate blrffer, pH 7.5, containing 0.6 mM XADH, and G PM ZnClz . The dilllted enzymes were incubated at room temperature for 2 hr and then dialyzed for 18 hr against 0.05 M phosphate bluffer, pH 7.5, at 4”. Controls Tere exposed to 0.1 M phosphate buffer, pH 7.5, instead of urea, then diluted, incubated, and dial)-zed in the same way as the reconst,ituted enzymes. Spectrophotometric data on reconstituted enzymes and controls were obtained on dialyzed solutions using a Cary 14 spectrophotometer at 1: 10 scale expansion. Enzy?ne assays. Enzyme assays were done in l-cm cuvettes at 340 rnp at 23.5” in 3 ml total volume, in one direct,ion using NAD, 0.45 mM; ethanol, 8.6 mnf; in glycine buffer, 0.062 M, pII 10.0, and in the opposite direction using NADII, 0.175 mnr; 5flDIIT, 0.11 mM; cyclohexanone, 0.11 mM; acetaldehyde, 1.2 mu, ill 0.03 M phosphate bluffer, pH 7.0.
DEHYDROGEXASE
259
E1ectrophore.si.s. The dilute, reconstituted enzymes and controls were concentrated by vacuum dialysis against 0.05 M phosphate bul‘fcr, pII i.5, to volumes rangitrg from 0.05 ml to 0.1 ml and adjusted by dilution to the same or similar activity/ml. Starch gels were made by adding 11.25 g starch (Connaught, Canada) per 100 ml of 0.025 M Tris HCl buffer, pH 8.5. Electrophoresis was done on horizontal starch gels at 4” for 15-20 hr at 200 V constalrt voltage. The gel dimensions were: 25.5 X 8.6 X 0.5 cm. In the electrode vessels 0.3 RI Tris HCl bufl’er, pH 8.5, was used. Cellulose acetate electrophoresis was carried out at roan, t,emperature in a Beckman apparatus at 0.175 mA constant, current per l-cm width of ~llulose acetate st,rip, for 2W.O min. Cellulose acetate was soaked in 0.025 M Tris IICl buffer, ~118.5, while the electrode butier was 0.05 1~. After completion of electrophoresis the following solution was Ilsed for activity staining of gels a,nd cellulose acetate strips: L)P?: 18 mg; NBT 10 mg; PMS 1 mg, ethanol ‘35:6 0.05 or 0.1 ml; So-OI-I-5p-IT-ketoandrost,an 2 mg per 25 ml of 0.05 M Tris HCl buffer, pH 8.5. When st,eroid was used alone as substrate, it was dissolved in 0.1 ml of acetone, when used together with ethanol, it was dissolved iI1 0.1 ml of ethanol. Starch gels, after slicing, were incubated in the above solutions at room temperatllre, in the dark, for varying lengths of time, depending on the reclllirement of the experiment and the initial load of t,he enzyme (15 min to 4 hr). Cellulose acetate strips were developed at room tempcrature, in the dark, by overlaying with another cellldose acetate strip soaked in the activit,y stain. For protein staining, nigrosin (obtained from (;. T. Gurr, London, England), 0.02cG w/v solution in 407, v/v ethanol in water containing 2.5’:; w/v trichloracetic acid, was r~scd for starch gels, and 0.002~~ w/v solution in 2(., v,:v acetic acid in water was used for cellulose acetate strips. Starch gels or cellulose acetate strips were placed in t,heir respective staining solutions and then left in closed boxes overnight. Excess stain was washed Cellldose away with 5X v/v acetic acid in water. acet)ate strips were also stained with Ponceau S, using stain and instrclctions supplied by Beckman, u. 8. A. Determination of the composition of isoenzymes after electrophoreais on cellulose acetate. Proteitl bands on cellulose acetate, visible after staining with ponceau S, were cut out and the adsorbed stain was eluted into l-ml vol of 0.1 N NaOII contaiued in IO-ml centrifrlge tribes. After the elution was complete, the bllle solutions were ceutrifuged t,o remove some celllllose acetate fibers. The amolmt of stain corresponding to variolls protein bands was determined colorimet-
a From b Values
(3 ,---_* L--.2
horse
Present in
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
commerf’a’ preparations
+
liver
Present in
SEPARATION
LADHs
LADHE
2
+
2
and ion-exchange of enzyme activity
+
f
+
f
+
2A
1
+
+
4 3
-I-
5
Ethanol activity
per
ss
ES
EE
in 3-ml
Nomenclature relating to subunit composition
-
ES’
ES”
EE’
EE”
composition
TsLW&i;
ISOIZNZYMES
per
mg enzyme
min
volume
SS’ 6,700h
130,000
250,000
Acetaldehyde
substrate
SEPARATED
SS” 4,900b
2,300
10-300
SODHT
with
OF THE
? 4,ooob
7-10,000
15 ) 500”
Ethanol
activity
PROPERTIES
Specific
SOME
?
?
I AND
chromatography3. = change of 0.001 OD
+
+
f
-
-
-
Steroid activity
TABLE ELECTROPHORESIS
y;ry;5cc!lan‘ Ref. 3
ON ZONE
Nomenclature as in Ref.
PATTERN
Identity confirmed by re-electrophoresis Identity not yet confirmed. One unit described in Materials and Methods. Ref. 2. from a single estimation.
<----. ..___, i---.,-,2
0
on zone electrophoresis
Isoenzymes of LADH
LADH
J
I
I
I
J }
in conditions
ss
ES
EE
Group subdivision
F
ii
8
G
$ u
t!
Ti K
cd E c3
HORSE LIVER ALCOHOL DEHYDROGENASE rically using at 540 mr.
a Beckman
1)U
RESULTS XOMENCLATURE OF LADH
spectrophotometer
ISOEN~YMES
LADH occurs in multiple isoenzymic forms (l-3). Some horse livers contain at least nine electrophoretically separable components (5) which migrate toward the negative electrode on starch gels at pH 5.5. A schematic representation of the electrophoretic separation pattern of LADH on solid supporting media is given in Table I. Since independent investigations on some of the LADH isoenzymes were carried out concurrently by two groups (1, 3) two different nomenclatures were used. They are presented in the table together with some properties of these isoenzymes and the proposed new nomenclature. In our previous paper (1) we showed that one of the isoenzymes consisted of two elec-
FIG. 1. Inactivation of ES in 8 M urea containing 0.1 M 2-mercaptoethanol in 0.1 M phosphate buffer, pH 7.5. The enzyme activity was assayed with 6.9 IIIM cyclohexanone. Other components of the assay system were as described in text. The control rate in 0.03 M phosphate buffer containing 0.133 M urea and 0.0016 M 2.mercaptoethanol was 23$& of that in the phosphate buffer alone, which indicates that urea and mercaptoethanol even in these low concentrations inhibit the reaction velocity.
291
trophoretically separable species of subunits. As a result of previous and present investigations we know that the more negatively charged subunit species (subunit E) is common to t,he hybrid isoenzyme (isoenzyme 1 or LADHs) and the homogeneous isoenzyme (isoenzyme 3 or L~DHE). The more positively charged subunit species (subunit S) is common to the hybrid isoenzyme and a homogeneous isoenzyme which exhibits even higher steroid activity than the hybrid. In the new nomenclature the hybrid isoenzyme, consisting of two differcnt subunits, is named ES, t,he homogeneous more negatively cha,rged isoenzyme, EE, and the homogeneous and more positively charged isoenzyme, SS. The use of E and S in the nomenclature for the other isoenzymes of LADH will be discussed. DISSOCIATION AND RECONGTITUTION OP EE, ES, AND 8s hxtivation of ES ia urea. ES (0.5 mg/ml) was dissolved in 8 M urea, 0.1 M phosphate buffer, pH 7.5, and 0.1 ~2-mercaptoethanol. Samples of 0.05 ml mere taken at different time intervals, and the enzyme activity was measured with cyclohexanone as substrate. The assay system contained 0.133 M urea and 0.00166 M 2-mercaptoethanol, in addition to the usual components described in Materials and Methods. The time course of the inactivation of ES is shown in Fig. 1. The results are expressed as percentage of the activity of the undissociated control. At times longer than S min the activity remained at a constant level of about 10% of the control, thus suggesting that t’he dissociation of ES may not be going to completion. Since in our experiments it was necessary that ES should be fully dissociated, 1y-e developed in collaboration wit’h Dr. J. Iioepke (6) an electrophoretic procedure by which dissociated and denatured J,ADH could be separated rapidly from the native undissociated enzyme. Using this technique we found that after 7 min exposure to S M urea containing 0.1 nr 2-mercaptoethanol ES, EE, and SS were no longer present in the solution. It is assumed, therefore, that the activity exhibited
292
PIETRUSZKO
AND
THEORELL
phoretic pattern of SS after dissociation and reconstitution. It is identical with the control SS in Fig. 2A. A similar comparison is made in Fig. 3B and 3A after EE was subject’ed to the same procedure. Fomnatiow of EE: and SS from ES. When pure ES was dissociated and reconstituted three enzymes were formed. In addition to ES, EE, and SS appeared. This is shown in Fig. 4, where the more negatively charged enzyme formed from ES is identified elec-
FIG. 2. Dissociation and reconstitlltion of SS. Starch gel after electrophoresis was stained for enzyme activity using a mixture of 5@DHT and ethanol as substrate. A, SS control, B, SS after dissociation and reconstit,rltion, C, ES after dissociation and reconstitution.
by the ES at times longer than 7 min is due to the partial reactivation of the enzyme during the assay. According to Hamburg’s (7) optical rotatory dispersion measurements on LADH (obtained from C. F. Boehringer & Soehne GmbH, Germany,) even shorter time (4 min) and less concentrated (7 M) urea were sufficient for complete dissociation. Dissociation and reconstitution of AS’Sand of EE. The results demonstrated in Figs. 2 and 3 show that SS and EE when subjected separately to t’he procedure of dissociation and reconstitution reappear with unchanged properties. Figure 2B shows the electro-
A
B
C
FIG. 3. l>issociation and reconstitution of EE. I, gel stained for enzyme activity using a mixture of @I)HT and ethanol as substrate, II, gel stained for protein with nigrosin. A, EE control, B, EE after dissociation and reconstitution, C, 50-8076 saturated ammonium sulfate fraction of horse liver extract, used as marker showing all isoenzymes of LAl)H except SS’ and SS”.
C
A
FIG. 4. Dissociation and reconstitution of ES. I, gel stained for enzyme activity using a mixture of 5pDHT and ethanol as substrate; II, gel stained for protein with nigrosin. A, ES control; B, ES after dissociat,ion and reconstitution; C, EE control.
HORSE
LI\-ER
ALCOHOL
DEHYDROGENASE
293
1: 1. The more negatively charged subunit of ES occurs exclusively in EE (Fig. GC), while SS consists exclusively of the more positively charged subunit (Fig. GA). DISSOCI~TIOK
OF
EE
ASD
I’KESENCE ASD ABSEXCE 2-l IEKC.\PT~ETH~~K~L
c FIN:. 5. Dissociation and reconstitkltioll of a misttIre of EJZ and SS Cellulose acetate after elect rophoresis was stained for enzyme artivit? with ethanol as substrat,e. A, mixture of BE and SS, colltrol; 13 and C. JXE and 88 after dissociation :tlld recollstiflltion; C, ES control.
t’rophoreticall;with corkol EE. The electrophoret,ic identification of the more positively charged enzyme, formed from ES, shown in Fig. 2. (Compare 2A and 2C). Forrti,ation of ES jr017~ a mixture of EE at/t/ Shy. In Fig. 5 (5B and 5C) the formation of Id% is demonstrated after subjection of a mixture of EE and SS (Fig. 3) to the dissociation and reconstitution procedure. ES, formed from the mixture of EE and SS is identified electrophoretically with the control ES (compare Figs. 5B and 5C with Fig. 51)). hkl~~opl~o~~esis of EE, ES, and SX in the dissociated state. EE, ES, and SS were exposed separately to X M urea in 0.1 M phosphate buffer, pH 7.5, containing 0.1 M 2-mcrcapt,oethanol and 0.01 M EDTA at room t~emperature for 1 hr. EDTA was used here to remove ZI?+ from the subunits (4) in order to avoid possible interference of bound Zn’f with the subunit electrophoresis. The electrophoretic patterns of dissociated El;,, ES, and SS arc shown in Fig. 6. Fig. GB shows the separation of two different, subunit.s of ES. The relative percentage composition of ES was determined colorimetrically after protein staining. In t,\vo experiments, \ve found 52 and 50% subunit’ E and 4S and 5OC; subunit S. ES consists, therefore, of E and S subunits in the ratio
ES
IX
THE
OF
Drum et al. (4) believed, on the basis of ultracentrifugation experiments, that LADH is dissociated into subunits of 40,000 RIW (“dime? ~20,~ = 2.4) or into subunits of 20,000 MW (“monomer” ~20,~ = 1.5) depending on the dissociation conditions. Electrophoresis of subunits oj EE and ES it2 “mo72omer” at/d “tlin7.er” cotditions. EE and ES, 10 mg/ml, were exposed to S M urea in 0.1 x phosphate buffer, pH 7.5, (“dime? conditions) (4) or in the additional presence of 0.1 M 2-mercaptoethanol and 0.01 M EDTA (“monomer” conditions with total Zn2+ removed) (4) at room temperature for at least 1 hr before electrophoresis. Dissociated enzymes were electrophoresed in S Y urea for “dime? conditions and in S I\/ urea containing 0.1 M 2-mercaptoethnnol for L‘n~onomer” conditions. Electrophoresis was carried out at room t’emperature at a constant current of 0.175 mA per cm width of cellulose acet’ate strip for 30 min in various buffer systems. The results of electrophoresis of dissociated EE and ES in the “monomer” and ‘Ldimer” conditions are shown in Table II. In both conditions EE migrates as one
+
-
ABC FIG. 6. Electrophoresis of E:E, ES, arld SS iu the dissociated form. Aft.er dissociation, electrophorzsis in 8 M tIrea containing 2.mercaptoethanol in glycille brlffer, pH 9.6 (for details see Table II), on celllllose acetate srlpporting mediums. A, SS; B. ES; C, EE stained with nirtusin.
294
PIETRUSZKO
AND TABLE
ELECTROPHORETIC
OF LADH
SEPARATION
s~~;i~;$~d cont. buffer (Ml
Separation
of LADH
subunits
Buffer added to 8 M urea and 0.1 M 2-mercaptoethanol Glycine NaOH Glycine NaOH Tris-HCl Sodium phosphate Sodium phosphate Sodium phosphate Sodium acetate Sodium acetate Separation
of LADH
subunits
Buffer added to 8 M urea alone Glycine NaOH Glycine NaOH Sodium phosphate Sodium phosphate Sodium phosphate
in “monomer”
0.025 0.025 0.025 0.0125 0.0125 0.0125 0.025 0.025
0.025 0.025 0.0125 0.0125 0.0125
@Mixture pH was measured on mercaptoethanol. b Migrate toward positive electrode,
IN
“MONOMER”
Sample applied near electrode + or -
Mixture PH’
AND
“DIMER”
Subunits distance migration (mm) E
CONDITWNS
Separation distance (mm)
S
Enzyrpes dissoclated (number of bands) EE
ES
conditions
0.05 0.05 0.05 0.025 0.025 0.025 0.05 0.05
in “dime?
II
SUBUNITS
.BuEer (M) In vessels electrode
THEORELL
-
10.5 9.5 8.9 7.8 7.8 7.0 5.8 4.8
11* 2b 4.5 5.0 2.5b 5.5 10 20.5
+ + + +
10” 1.5 7.5 6.5 1.25b 7.0 10 20.5
1.0 3.5 3.0 1.5 1.25 1.5 0 0
1 1 1 1 1 1 1 1
2 2 2 2 2 2 1 1
0 4.5 4.5 8.0 10.0
2.5 3.0 1.0 1.25 1.25
1 1 1 1 1
2 2 2 2 2
urea,
and/or
conditions
0.05 0.05 0.025 0.025 0.025 pH
-
10.17 9.57 8.5 7.67 6.6 meter
all others
after
+ + + mixing
not marked
band and ES as two bands, except for lower pH values where E and S subunits are not separable. Since in the “dime? conditions Zn2+ was not to be removed from the subunits (according to Drum et al. (4) removal of Zn2+ results in a further dissociation of the “dime?‘), the electrophoresis had to be confined to the pH values at which no loss of Zn2+ from the LADH molecule was likely to occur. Although the rate of electrophoretic migration of the subunits obtained in “dime? and “monomer” conditions is slightly different, the degree of separation obtained in buffers of comparable pH values is almost identical. The small variations in the absolute rates of migration are very probably due to technical imperfections. Since the “monomeric” subunits had their Zn2+ completely removed by EDTA during dissociation (4), while the “dimeric” ones
2.5b 1.5 3.5 6.5 8.5
-
buffers
migrate
toward
with
8 M negative
2-
electrode.
had all their Zn2+ intact (4), we attempted a separation of the EE “monomers” and “dimers” by simultaneous electrophoresis. Thus, EE was dissociated in two batches: one in “monomer” and another in “dime?’ conditions. Both were then electrophoresed in several buffers containing 8 M urea. No difference in the rate of migration of these subunits was observed. An attempt was also made to separate “monomers” and “dimers” of EE by chromatography on Sephadex G-150 and G-200 columns but the void volumes with “monomers” were the same as those with “dimers” (6). Reconstitution of ES after dissociation in “dime? conditions. After dissociation in the absence of 2-mercaptoethanol, ES was reconstituted by dilution with phosphate buffer containing NADH (0.6 mM) and 2-mercaptoethanol, (0.01 M) (the latter was
HORSE TABLE EFFECT THE
0 8 15 :
ALCOHOL
III
OF DISSOCIATION RECONSTITUTION Substrates
Dissociation time (minj
LIVER
TIME
ON
OF ES (% reconstitution)
Acetaldehyde
Cyclohexanone
Ethano,
100 30.5 26.8 22.8 25.3 22.2
100 27.7 24 24 23.2 20.2
100 25.5 24.7 23 23 17.3
59 D HT
100 38 32.4 35.4 31.8
+
5432112345 Fro. 7. Effect of dissociation time on the isoenzyme composition resulting from combination of E :uld S subunits. I, starch gel stained for erlzymr artivity with a mixture of 5pDHT and ethanol :M substrate; II, stained for protein with nigrosin. 1, control before dissociation; 2, after 8 mill dissociation; 3, 15 min; 4, 30 min; 5,45 min.
used to protect the reconstituted enzymes against SH oxidation). After incubation, dialysis, concentration, and electrophoresis, formation of EE, ES, and SS was demonskated by protein and activity staining. The result was indistinguishable from that in “monomer” conditions (see Fig. 2). Efect of’ dissociation time on 7.econstitution o.f E’S. ES was subjected to the dissociating procedure in “monomer” conditions and different batches were diluted after different t,imes with a solution containing phosphate, NADH and ZnClz. After incubation followed by dialysis, the percentage of regain of the enzyme activity was determined spectrophotometrically, and Dhe isoenzyme composition was analyzed by electrophoresis.
DEHTDROGENASE
295
The effect of dissociation time on the regain of ethanol-, cyclohexanone-, acetaldehydeand 5@DHT-activity is shown in Table III and the effect of the dissociation time on t,he composition of isoenzymes in Fig. 7. The percentage regain of various activities of LADH isoenzymes is only slightly dependent on the dissociation times between S-60 min, and the composition of isoenzymes (Fig. 7) appears to be independent of the time of dissociation. E,J’ect of temperature on the reconstitution of ES. ES was dissociated at room temperature in “monomer” conditions. One sample m-as reconstituted by dilut,ion at 23” and another at 3S”. The reactivation, as measured by activity with cyclohexanone, was found to be 4O’X at 23” and 35 % at 3s” and in both cases only EE, ES, and SS were formed. E$ect of Zn2+ concentration of the reconstitution of E;S. ES (0.5 mg/ml) was dissociated conditions and then reconin ‘5nonomer” stituted by dilution with phosphate buffer, containing NADH and varying amounts of ZnClz. The effect of Zn2+ on the reconstitution of ES is shown in Fig. 8 and Table IV. Electrophoretic analysis at’ different concentrations of added Zn*+ (Fig. 8) shows that the increase of Zn2+ depresses t’he re-
FIG. 8. Ilccotlstittttion of ES at differetlt concentrations of Zn*+. Starch gel after electrophoresis was stained for enzyme activity using a mixture of 5pDHT and ethanol. Iteconstitkttion at different concentrations of Zn2+. 1 no Zn”+ added; 2. (i PM; 3, 18 ,.IM; -1-, 54 PM; 5, l& PM. The tmdissociated u)ntrol was the same as ill Fig. 7.
296
PIETRUSZKO TABLE
AND
IV
EFFECT ON ZN~+ ON THE RECONSTITUTION OF ES Percentage Percentage Ratio of ZnClz added reconstitution reconstitution SPDHT to during reconstitu- basedon cyclohexation (MM) cyclohexanone %A? none activity activity activity 0.0 6.0 18.0 54.0 162.0
Cont,rol
38.7 30.8 16.6 8.9 8.6 100
51.3 52.7 39.0 14.5 13.7 100
5.5 7.2 9.8 G.8 G.G 4.2
constitution of EE more than that of SS. The ratio of the activities with 5PDHT resp. cyclohexanone shows a maximum at 18 &!l Zn2+. The greatest regain of enzyme activity is obtained when no Zn2+ is added. In our experimental conditions 6 JVl Zn2+ inhibits the regain of the cyclohexanone activity and affects the composition of the isoenzymes formed from E and S subunits. Thus, with 6 PM Zn2+, EE: ES :SS was 1: 7 :2 while with no Zn2+ added, it was 1: 2: 1, as expected for simple statistical combination of two different subunits. Dissociation of ES in the presence of d-mercaptoethanol and EDTA. ES was dissociated in “monomer” conditions in the presence of 0.01 M EDTA (4). Under these conditions the E and S subunits lost their ability to recombine and neither ethanol nor steroid activity appeared on dilution. Electrophoresis showed that all the enzyme was present in solution in the denatured form and that no reassociation had occurred. This is in agreement with the results obtained by Drum et al. (4) on Boehringer LADH (mainly EE). DISCUSSION
In 1951 Theorell and Bonnichsen (8) established that each LADH molecule has two coenzyme-binding sites. Kinetic work carried out later in this laboratory strongly suggested that these two sites are functionally separate, equivalent, and independent (9). These studies were done on LADH which consisted predominently of EE. The evidence for two coenzyme-binding sites on ES (LADHs) was presented by
THEORELL
Theorell et al. (2) in 1966. Simultaneous investigations carried out in two laboratories (2, 3) demonstrated that the steroid activity of crystalline LADH was confined to ES and that EE, the major component, was almost devoid of this activity. Kinetic experiments with 3~OH cholanic acid used as a competitive inhibitor (a), or using mixtures of two substrates and by alternate product inhibition (3) showed that the reaction of steroids and classical substrates of LADH occurred at different catalytic sites. Work with antibodies (10, 11) estabblished that the two catalytic sites were located on t’he same protein molecule (ES) and indicated close structural relat’ionship between EE and ES. Further work (1) showed that EE and ES had a subunit in common (subunit E) and also that ES contained another subunit (subunit S) which was absent in EE. Present studies show that one of the naturally occurring isoenzymes of LADH (5) consists exclusively of these S subunits. Three isoenzymes of LADH (EE, ES, and SS) are formed by the hybridization of E and S subunits. ES is a hybrid consisting of equal amounts of these subunits. EE consists only of subunit#s E and SS of subunits S. Fluorimetric data of Theorell et al. (1968) (12) show that in the SS molecule there are also two catalytic sites which are equivalent with one another but different from those in EE. In our previous paper (1) we suggested that the steroid activity of LADH was associated with subunit S and ethanol activity with subunit E. This conclusion was derived from the study of the then available ES. Since small activity with nonsteroid substrates of the S site of this enzyme was obscured by the very high activity of its E site, it was thought t’hat the S site of ES was only active with steroids. However, later studies, especially after isolation of SS, have shown that although significant activity toward steroids is exhibited only by the isoenzymes containing subunit S, all LADH isoenzymes (including SS) are active with ethanol. The preliminary data on specific activities of EE, ES, and SS, obtained with 5PDHT, acetaldehyde, and ethanol, presented in Table I alongside with subunit composition, demonstrate
HORSE
LIVER
ALCOHOL
structure-activity relationship of these three isoenzymes. EE has high activity with classical LADH substrates, and only negligible, if any, activity with steroids. The activity of EE with ethanol and acetaldehyde is about twice that of ES. Although still active with ethanol, SS has a steroid activity which is twice tha,t of ES and very low activity with acetaldehyde. The activity of SS with acetaldehyde was in fact so low that it could have been caused by a small amount of EE or ES impurity. Extended kinetic experiments are required in order to clarify this interesting difference. The catalytic properties of ES are intermediate between those of EE and SS. This is in complete agreement with the subunit composition, since EE consists exclusively of subunits E, and SS of subunits S, while ES is a 1: 1 hybrid of these subunits. Since only SS and ES exhibit significant activity with the steroid substrates, the catalytic site concerned with the interconversion of steroids must be located on subunit S. The catalytic site concerned with the classical LADH substrates, but not with steroids, is present in subunit E. Chemical data on LADH, such as peptide mapping (13, 14)) end group determination (7, 15), X-ray data (16), and the fact that only two NADH molecules are bound per molecule of the enzyme (8), indicate that LADH is composed of two subunits. Further dissociation of LADH as reported by Drum et al. (4) cannot be reconciled with our evidence. After hybridization of E and S subunits, after dissociation of ES in Drum et al. (4) “monomer” and “dimer” conditions, we find only three LADH isoenzymes: homogeneous EE, hybrid ES, and homogeneous SS. If the quaternary structure of an enzyme is tetrameric and the reconstitution is random, five isoenzymes are expected from combination of two kinds of nonidentical subunits (17), if it is dimeric, only three enzymes can be obtained. Our attempts to show different subunits (6) in “monomer” and “dime? conditions (4) were unsuccessful (see above). Moreover, it is known that EE contains 28 cysteine residues per molecule (lS), 8 tyrosine residues, and 4 tryptophane residues (2).
297
DEHYDROGENASE
Recently Jornvall and Harris (1968) (19) in a study of tryptic digests isolated 14 unique cysteine peptides, 4 unique tyrosine peptides, and 2 unique tryptophane peptides per molecule of EE. This is expected for two identical 40,000 MW “dimers” but incompatible with a structure of four identical 20,000 MW “monomers.” In the recent Alfred Benzon Symposium in Copenhagen (10/g/68) (20) Dr. Howard Schachman showed new ultracentrifugation data on LADH demonstrating that the sedimentation constants under ‘Lmonomer” and “dime? conditions were the same, with values intermediate between those of Drum et al. (4) ~20,~ = 1.5 and 2.4. Prom the above considerations it appears that E and S subunits in both “monomer” and “dime? conditions represent halves of the LADH molecule. The combination of E and S subunits accounts for the structure of only three isoenzymes of LADH of the nine (5) present in horse liver (Table I). Since attempts to form isoenzymes other than EE, ES, and SS by the combination of E and S subunits have been unsuccessful, it appears that other subunit species must be involved in order to explain the composition of the remaining LADH isoenzymes. LADH separates on electrophoresis in a repeated pattern of three isoenzymes in three groups (5). EE, ES, and SS are the most positively charged in each group (Table I) and occur as the major components. As the structure of isoenzymes other than EE, ES, and SS is at present unknown, but the preliminary activity data suggest greater similarity between those in a given group than between individual members of separate groups, we have named these isoenzymes by attributing to them a structure similar to that of the known isoenzyme in the group. Thus, the isoenzyme migrating on electrophoresis just slower than isoenzyme EE is called EE’ and the one even slower EE”. Other isoenzymes of LADH have been named in an analogous way. REFERENCES IL, RINGOLD, H. J., LI, T-H., 13. L., .&U;SON, A., AND THEORELL, Nature 221,440 (I%%).
1. PIICTRUSZICO, VALLXE,
H.,
298
PIETRUSZKO
2. THEORF,LL, H., TANIGUCHI, S., AKESON, A,. AND SKURSKP, L., Biochem. Biophys. Res. Commun. 24, 603 (1966). 3. PIETRUSZKO, R., CLARK, A. F., GRAVES, J. M. H., AND RINGOLD, H. J., Biochem. Biophys. Res. Commun. 23, 526 (1966). 4. DRUM, D. E., HARRISON, J. H., LI, T-K., BETHUYE, J. L., AND VALLEE, B. L., Proc. Natl. Acad. Sci., U. S. 67,1434 (1967). 5. THEORELL et al. Unpublished data. 6. KOEPKE, J., WKESON, A., AND PIETRUSZKO, R., (1969) to be published. 7. HAMBURG, R. D., The Physical Properties of Horse Liver Alcohol Dehydrogenase. (Diss.) University of California, Berkeley (1966). 8. THEORELL, H., AND BONNICHSEN, R. K., Acta Chem. Stand. 6, 1105 (1951). 9. THEORELL, H., Harvey Lectures Ser. 61, 17, (1967). 10. PIETRUSZI~O, R., AND RINGOLD, H. J., Biochem. Biophys. Res. Commun. 33,497 (1968).
AND
THEORELL
11. PIETRUSZKO, R., RINGOLD, H. J., KAPLAN, N. O., AND EVERSE, J., Biochem. Biophys. Res. Commun. 33, 503 (1968). 12. THEORELL, H., AKESON, A., LISZKA, B. M., AND DF, ZALENSKI, C. W. T., (1969). Manuscript in preparation. 13. HARRIS, J. I., Nature 203,30 (1964). 14. LI, T-K., AND VALLE, B. L., Biochemistry 3, 869 (1964). 15. JBRNVALL, H., Acta Chem. Stand. 21, 1805 (1967). 16. BRAND&N, C. I., Arch. Biochem. Biophys. 112, 215 (1965). 17. MARKGRT, C. L., Science 140,1329 (1963). 18. YONETANI, T., AND THEORELL, H., Arch. Biochem. Biophys. 99, 433 (1962). 19. JBRNVALL, H., AND HARRIS, J. I., Abstract 769 FEBS Fifth Meeting, Praha (1968). 20. SCHACHMAN, H., Alfred Benson Symposium, Copenhagen (1968).