ANALYTICAL BIOCHEMISTRY 69, 40 I-409 ( 1975)

Preparative Purification of Homogeneous

Steroid-Active lsozyme of Horse Liver Alcohol

Dehydrogenase by Affinity Chromatography

on an Immobilized AMP-Analog

LARS ANDERSSON,~ HANS J~RNVALL,’ AND KLAUS MOSBACH~

1 Biochemical Division, Chemical Center, University of Lund, P.O. Box 740, S-220 07 Lund 7, Sweden, and “Department of Chemistry,

Karolinska lnstitutet, S-104 01 Stockholm 60, Sweden

Received March 31, 1975: accepted June 24, 1975

The homogeneous steroid-active isozyme, SS, of horse liver alcohol dehy- drogenase was purified preparatively from a horse liver crude extract by affinity chromatography on W-(6-aminohexyl)-AMP-substituted Sepharose after a batch- wise prepurification on CM-cellulose. The enzyme obtained showed only one band on agarose-gel electrophoresis and on dodecyl sulphate-gel electrophoresis: the yield was about 30 mg SS isolated from 260 g horse liver.

The dimeric enzyme horse liver alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) has three isozyme forms known as EE, ES, and SS (1) (or AA, AB. and BB (2)) due to synthesis of two dif- ferent types of protein chains (3). In addition, further multiplicities are known since subfractions, related to each of the three isozyme forms, may occur (4, 5) as well as a possible polymorphism in the SS isozyme (6). The isozymes differ in substrate specificity. All three are active toward ethanol as substrate, but only the ES and SS forms are active toward 3p-hydroxysteroids (7-9).

The main isozymes have already been separated and purified by con- ventional methods (4-6, 9-l l), which, however, are time-consuming and often result in low yields. In the present work, therefore, affinity chromatography has been studied as an alternative purification method. It was recently reported (12) that, in a mixture of isozymes, the EE and SS forms may be separated bv affinity chromatography on a column coupled with the general ligand AMP (13). In the procedure described here a method has been developed to purify the SS isozyme on a preparative scale from horse liver crude extracts. Affinity chroma- tography on N6-(6-aminohexyb-AMP-substituted Sepharose is used in combination with a batchwise prepurification step on CM-cellulose and an electrophoretically pure isozyme is obtained.

401

Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

402 ANDERSSON, JijRNVALL AND MOSBACH

MATERIALS AND METHODS

Materials

Cholic acid (sodium salt), glutathione (reduced form, crystalline), /3-NAD+ (grade III), /3-NADH (grade III), and Sp-dihydrotestosterone (Sp-DHT) were obtained from Sigma (St. Louis, MO.). Sepharose 4B and Sephadex G-25 (medium) were bought from Pharmacia (Uppsala, Sweden) and CM-cellulose (CM 52) from Whatman (Maidstone, Eng- land). Acrylamide, N,N’-methylenebisacrylamide and N,N,N’,N’-te- tramethylethylenediamine were obtained from Eastman (Rochester, N.Y.) and Coomassie brilliant blue and sodium dodecyl sulphate from Schwarz/Mann (Orangeburg, N.Y.). All other chemicals were of analyti- cal grade and were used without further purification.

AMP-Sepharose Preparation

AMP-Sepharose was prepared by coupling N6-(6+uninohexyl)- AMP to Sepharose (14), utilizing the BrCN procedure (15). The amount of bound nucleotide was determined from the phosphate con- tent, which was analyzed after total combustion of freeze-dried gel (16). Approximately 40 pmoles of AMP were bound per gram of dry gel.

Analytical Procedures

Alcohol dehydrogenase activity toward ethanol was measured as pre- viously described (12). Alcohol dehydrogenase activity toward S/3-DHT was measured by following the decrease in absorbance at 340 nm after the addition of NADH (0.14 pmoles), 5@-DHT (0.17 pmoles) dissolved in 25 ,ul of acetone, and the test solution (5-50 ~1) to 0.03 M sodium phosphate, pH 7.0. The final volume of the incubation mixture was 3 ml.

Isozymes were identified by agarose-gel electrophoresis (12) and en- zyme preparations were also analyzed by sodium dodecyl sulphate elec- trophoresis (17).

Protein was determined according to Lowry et al. (18) with serum al- bumin as standard, and corrections were made for interference from cholic acid, NAD+, NADH, and glutathione.

Pretreatment of Extract

All procedures were performed at 4°C. A frozen horse liver was thawed, ground in a meat grinder, and extracted overnight with 2 vol of 0.01 M sodium phosphate, pH 7.5, under continuous stirring. After filtra- tion through cheesecloth, centrifugation (23,OOOg, 4 hr), and filtration through glass wool the extract was dialyzed for 20 hr with one change against a tenfold volume of 0.01 M sodium phosphate, pH 7.5. To this solution 200 g of preswollen CM-cellulose equilibrated with 0.01 M

PURIFICATION OF STEROID-ACTIVE ADH 403

sodium phosphate, pH 7.5, was added. The mixture was stirred for 2 hr and then centrifuged (lO,OOOg, 10 min). The supernatant was discarded and the cellulose washed with equilibration buffer. After centrifugation the resin was extracted with 500 ml of 0.1 M sodium phosphate, pH 7.5, for 2 hr under continuous stirring. The mixture was centrifuged (lO,OOOg, 10 min) and the supernatant collected. The resin was then washed with 0.1 M sodium phosphate, pH 7.5. The supernatants were mixed and solid ammonium sulphate was added with stirring to 70% sat- uration in order to concentrate the protein solution. The pH was kept at about 7-8 by addition of ammonia. The solution was stirred occasionally for 2 hr and then centrifuged (1 S,OOOg, 1 hr). The supernatant was dis- carded and the precipitate dissolved in 0.1 M sodium phosphate, pH 7.5, 1 mM in glutathione, and dialyzed overnight against a tenfold volume of the same buffer. The solution was then centrifuged (39,OOOg, 1 hr) to remove undissolved material and the resulting clear orange solution ( 150 ml containing approx 900 mg protein) was subsequently subjected to af- finity chromatography. The total number of units of ethanol dehy- drogenase activity was about 150.

Afinity Chromatography

All chromatographic procedures were performed at 4°C. The affinity column (1.5 X 15 cm) was packed with approximately 20 ml of AMP- Sepharose and was equilibrated before each experiment with a few vol- umes of 0.1 M sodium phosphate, pH 7.5, 1 mM in glutathione. The alcohol dehydrogenase solution from the previous step was applied and the column then washed with about 10 vol of equilibration buffer and, when appropriate, with approximately 6 vol of 0.2 mM NAD+. Finally the enzyme was eluted with 1.5 mM cholic acid plus 0.2 mM NAD+.

RESULTS AND DISCUSSION

Initially, it was attempted to purify the SS isozyme of alcohol de- hydrogenase directly from a horse liver crude extract by affinity chroma- tography. Only partial purification was, however, obtained in a single step procedure, using a gradient of NAD+ in the presence of cholic acid. Elution was achieved by ternary complex formation of enzyme, NAD+ and the steroid inhibitor cholic acid. The SS isozyme, which has the greatest activity toward steroids, formed the strongest ternary com- plex and was therefore eluted first, and enriched in the fractions of the first peak (Fig. 1). This method is known to separate the homogeneous isozymes (12) but the poor purification of the three isozymes from the liver crude extract is probably due to the large number of other enzymes present which compete with the Sepharose-bound AMP-analog. This is not surprising, since AMP is an inhibitor for many enzymes, and this

404 ANDERSON, JiiRNVALL AND MOSBACH

orrgin - - + A B C

FIG. 1. Separation of the isozymes of alcohol dehydrogenase from a horse liver crude extract on an AMP-Sepharose column by elution with NAD+ plus cholic acid. (a) Elution curve from the column (1 X 14 cm, containing 9.6 ml wet gel) loaded with 7 ml of a crude extract containing 340 mg of protein and 26 units of ethanol dehydrogenase activity. Prior to the elution with a gradient of NAD+ (O-8 mM, 1.5 mM in cholic acid, total volume 200 ml) the column was washed with 5 vol of 0.1 M sodium phosphate, pH 7.5, 1 mM in glu- tathione followed by 3 vol of the same solution but also 1.5 mM in cholic acid. Fractions of 0.8 ml were collected at a flow rate of 3.0 ml/hr. (@-a), Horse liver ethanol dehy- drogenase activity. (b) Agarose-gel electrophoresis in 0.05 M Tris-HCl, pH 8.5, followed by staining for enzymatic activity. Hatched areas indicate less intense bands and dotted outlines hardly visible bands. The relative intensity of the bands is somewhat variable depending on different concentrations of the samples. Even the pattern of bands may vary slightly due to formation of subfractions. This can, however, be depressed by the presence of thiols. Forms just below main isozymes are subfractions and the letters refer to the hatched areas in (a).

property has also recently been utilized in a number of studies using such analogs in so-called general ligand atfinity chromatography ( 19, 20).

The problem of competing enzymes and the resulting reduced capac- ity of the affinity adsorbent when crude extracts are applied can be over- come by a pretreatment to lower the amount of other proteins. Batchwise fractionation on CM-cellulose prior to the affinity chroma- tography was therefore tested. A crude extract from 260 g of horse liver, partially purified on CM-cellulose, was applied to a column of W-(6-

PURIFICATION OF STEROID-ACTIVE ADH 405

FIG. 2. Affinity chromatography of the SS isozyme of alcohol dehydrogenase from a CM-cellulose pretreated horse liver crude extract on an AMP-Sepharose column (1.5 X 15 cm, containing 22 ml wet gel). Elution with 0.1 M sodium phosphate, pH 7.5, 1 mM in ghr- tathione, the same solution containing cholic acid plus NAD+ (1.5 and 0.2 mM, respec- tively) or NADH (10 mM) was started as indicated by the arrows. Fractions of 3.7 ml were collected at a flow rate of 7.2 ml/hr. (e-a), Horse liver ethanol dehydrogenase activity; (n--A), protein concentration.

A FIG. 3. (a) Dodecyl sulphate-gel electrophoresis at pH 7.3. (b) Agarose-gel elec-

trophoresis at pH 8.5. (A), Horse liver crude extract; (B), protein not adsorbed onto the CM-cellulose; (C), protein applied to the affinity column after the pretreatment step: (D), protein from pooled fractions of hatched peak in Fig. 2. Details of agarose-gel elec- trophoresis and band intensity are as for Fig. lb.

a 1 b

origin - - - -

+ + A B C D

A c D

406 ANDERSON, JtiRNVALL AND MOSBACH

aminohexyl)-AMP-substituted Sepharose. The elution profile is shown in Fig. 2. After extensive washing with buffer the SS isozyme was eluted with 1.5 mM cholic acid plus 0.2 mM NAD+; 1.5 mM cholic acid alone did not elute any isozyme. Fractions were pooled (hatched area in Fig. 2) and the protein was tested for purity by agarose-gel electrophoresis and dodecyi sulphate-gel electrophoresis. The pictures obtained (Fig. 3) reveal that the affinity step resulted in a preparation that was highly enriched in isozyme SS but still contaminated with other isozymes and proteins. The partly purified solution was therefore rechromatographed on the same column of AMP-Sepharose after regeneration of the column with a 5-ml pulse of 10 mM NADH (Fig. 2). However, prior to applica- tion the solution was freed from cholic acid and NAD+ by gel filtration through a column of Sephadex G-25. The elution profile of the rechro- matography step is shown in Fig. 4a. Weakly bound nucleotide depen- dent proteins other than alcohol dehydrogenase were removed at this stage with a wash of 0.2 mM NAD+3 and the SS isozyme subsequently eluted with cholic acid plus NAD+, as before. The SS isozyme was now obtained completely pure as judged by agarose-gel electrophoresis and dodecyl sulphate-gel electrophoresis (Fig. 4b).

Table 1 summarizes the results of the purification procedure. The whole bulk of SS- and some of the EE- and ES-content was adsorbed onto the CM-cellulose at pH 7.5 in 0.1 M phosphate (Fig. 3), whereas 95% of the total protein content did not stick to the resin under these conditions. The yield of 18% and the purification factor of 143 is deter- mined from activity measurements with SP-DHT as substrate, but as the ES form present in the horse liver crude extract is also active toward this steroid, the true yield of the SS isozyme should be even larger. The relative values of purification factor and yield appear considerably lower when enzyme activity against ethanol as substrate is taken as reference since the EE isozyme is present in vast excess in the original extract. These figures therefore do not apply directly to any isozyme. The total amount of SS per kilogram of horse liver can be calculated to be about 110 mg; by comparison about 200-600 mg of EE have been obtained by conventional isolation techniques (9). The present recovery is larger than that reported for the SS isozyme by ion-exchange chromatography when 79 mg/kg horse liver was obtained (5). The yield was then 0.2% when activity was measured with ethanol as substrate compared to 1.4% in the present work.

Affinity chromatography has been used for the isolation of many en-

3 Such a “NAD+-wash” in the preceding affinity step (Fig. 2) would have resulted in appreciable loss of alcohol dehydrogenase activity due to its weak binding in the presence of many other competing enzymes.

PURIFICATION OF STEROID-ACTIVE ADH

b - -

origin -

407

FIG. 4. Affinity chromatography of the SS isozyme of horse liver alcohol dehydrogenase on an AMP-Sepharose column ( 1.5 X 15 cm, containing 22 ml wet gel). (a) Rechroma- tography of pooled fractions of hatched peak in Fig. 2. Elution with 0.1 M sodium phosphate, pH 7.5, 1 mM in glutathione, the same solution containing NAD+ (0.2 mM) or cholic acid plus NAD+ (1.5 and 0.2 mM, respectively) was started as indicated by the arrows. Fractions of 3.7 ml were collected at a flow rate of 7.2 ml/hr. (0-O). Horse liver ethanol dehydrogenase activity; (n--n), protein concentration. (b) Electrophoreses on pooled fractions of hatched peak in (a). (A), Dodecyl sulphate-gel electrophoresis at pH 7.3; (B), agarose-gel electrophoresis in 0.05 M Tris-HCl, pH 8.5. followed by staining for enzyme activity.

zymes (2 1) and for purifications of some dehydrogenases from crude systems (22,23). It is now also evident that isozymes of dehydrogenases may be purified on a preparative scale from crude extracts by affinity chromatography as reported for alcohol dehydrogenase (this work) and lactate dehydrogenase (23, 24). In the preparation a prepurification step using batchwise chromatography on CM-cellulose has been added in order to utilize the capacity of the affinity adsorbent more efficiently. The present method is convenient and leads to homogeneous SS iso- zyme within a few days.

TABL

E 1

PURI

FICA

TION

OF

HO

MOG

ENEO

US

STER

OID

-ACT

IVE

ISOZ

YME

OF

HORS

E LI

VER

ALCO

HOL

DEHY

DROG

ENAS

E BY

AF

FINI

TY

CHRO

MAT

OGRA

PHY

ON

A SE

PHAR

OSE-

BOUN

D AM

P-AN

ALOG

Y zi E

Tota

l Sp

ecifi

c Eu

rifi-

%

Enzy

me

activ

ity

prot

ein

activ

ity

catio

n Yi

eld

0 St

ep

(uni

ts/m

l) (to

tal

units

) (w

) (u

nits

/mg)

(n

-fold

) (%

) z 5:

Cr

ude

extra

ct

0.67

(4.

5)

321

(216

0)

23,0

00

0.01

4 (0

.09)

1

(1)

loO

(100

) 4

CM-c

ellu

lose

0.

13 (

1.6)

11

5 (1

46)

1110

0.

10

(0.1

3)

7.1

(1.4

) 36

(6.

8)

Amm

oniu

m

sulp

hate

2

prec

ipita

tion

0.67

(0.

89)

108

(144

) 93

0 0.

12

(0.1

5)

8.6

(1.6

) 34

(6.

7)

F Fi

rst

affin

ity c

hrom

atog

raph

y 1.

0 (0

.62)

71

(43

) 40

1.

8 (1

.1)

129

(11)

22

(2.

0)

Seco

nd a

ffini

ty c

hrom

atog

- fs

w

hy

0.84

(0.

46)

58 (

31)

29

2.0

(1.1

) 14

3 (1

1)

18 (1

.4)

%

n Th

e fir

st f

igur

es f

or s

peci

fic a

ctiv

ity,

purif

icat

ion,

an

d yi

eld

appl

y to

alco

hol

dehy

drog

enas

e ac

tivity

te

sted

ag

ains

t 5/

3-D

HT

as s

ubst

rate

in

z

orde

r to

est

imat

e va

lues

mos

t cl

osel

y re

late

d to

the

SS

isoz

yme.

Du

e to

the

pre

senc

e of

the

ES

isoz

yme

in t

he c

rude

ex

tract

th

e va

lue

for

the

b yi

eld

is s

till

a m

inim

um

valu

e.

Figu

res

for

alco

hol

dehy

drog

enas

e ac

tivity

te

sted

ag

ains

t et

hano

l as

sub

stra

te

are

give

n wi

thin

pa

rent

hese

s an

d T

give

litt

le

indi

catio

n ab

out

purif

icat

ion

or y

ield

of

the

SS i

sozy

me.

PURIFICATION OF STEROID-ACTIVE ADH 409

ACKNOWLEDGMENTS

The authors thank Margaretha Scott for drawing the diagrams, Dr. P.-O. Larsson for synthesizing the AMP-analog, and Dr. Richard Venn for linguistic advice. The present in- vestigation has been supported by Statens Naturvetenskapliga (Project No. 2616-019), and Medicinska Forskningsrad (Project No. 13X-3532), and Kungliga Fysiografiska Salk+ kapet.

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