Purification and chemical characterization of alanine dehydrogenase of Bacillus subtilis

BIOCHIMICAET BIOPHYSICAACTA

33

BBA 65084 PURIFICATION AND CHEMICAL CHARACTERIZATION OF ALANINE DEHYDROGENASE OF BACILLUS SUBTILIS AKIRA YOSHIDA AND ERNST FREESE Laboratory of Molecular Biology, National Institutes of Health, National Institute of Neurological Diseases and Blindness, Bethesda, Md. (U.S.A.)

(Received March Ioth, 1964)

SUMMARY I. Bacillus subtilis alanine dehydrogenase (L-alanine: NAD oxidoreductase (deaminating), EC 1.4.1.1. ) has been purified by column chromatography using calcium phosphate gel, DEAE-Sephadex and Ecteola-cellulose; it has been obtained in crystalline form. 2. The sedimentation pattern indicated a homogenous preparation. The sedimentation constant at infinite dilution was 8.8 S. The molecular weight estimated by the sedimentation equilibrium method was 228 ooo. 3- Amino acid analysis gave the following ratios of amino acid residues: Asp, 43; Thr, 34; Ser, 2o; Glu, 51; Pro, 25; Gly, 51; Ala, 65; CySH, 2; Val, 49; Met, I I ; iLeu, 28; Leu, 42; Tyr, 15; Phe, 7; Try, I; Lys, 28; His, 9; Arg, 14.

INTRODUCTION Dehydrogenases t h a t require NAD or N A D P as cofactor presumably are related to one another by evolution. They are furthermore subject to various control mechanisms, most of which involve the enzyme itself or some precursor of it. Both relatedness and control can be analyzed at the molecular level only when the molecular properties of the corresponding enzymes are known. We have therefore started the isolation and characterization of some dehydrogenases in Bacillus subtilis. This organism was selected for three reasons. First, various mutants, particularly mutants involved in control mechanism, can be isolated and characterized genetically. Second, bacteria can be grown under derepressed conditions, which makes available a starting material already containing a few percent of a given dehydrogenase. Third, control mechanisms of enzyme formation and function can be associated with the properties of differentiation exhibited by sporulation and germination. In this paper, we show t h a t alanine dehydrogenase (L-alanine:NAD oxidoreductase (deaminating), EC 1.4.1.I), lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) and malate dehydrogenase (L-malate:NAD oxidoreductase, EC 1.1.1.37 ) can be separated from each other by column chromatography, although they are all large enzymes. We have crystallized alanine dehydrogenase and determined both its molecular weight and its amino acid composition. Biochim. Biophys. Aeta, 92 (1964) 33-43

34

A. Y'OSHIDA,E. FREESE

Alanine dehydrogenase has been first described, partially purified from B. subtilis and enzymatically characterized by WIAME AND PI~RARD 1,2. In spores and vegetative cells of B. cereus, a similar enzyme has been found, which has been partially purified and whose enzymic properties have been analysed in more detail3, 4. The synthesis of alanine dehydrogenase in vegetative cells of B. subtilis can be induced by alanine 5 and derepressed by nicotinic acid starvation 6.

MATERIALS AND METHODS

B. subtilis, Strain 6o127 (nicotinic acid requiring). Ion exchanger: Ecteola-cellulose from Serva Entwicklungs Labor, Heidelberg; DEAE-Sephadex from Pharmacia Fine Chemicals, Inc., Uppsala. Calcium phosphate gel: Hydroxyl apatite was prepared b y the method of TISELIUS, HJERT~N AI~D LEVIN 7. Culture medium: minimalglucose medium 8 supplemented with DL-alanine (o.I mg/ml) and nicotinic acid (0.03/~g/ml). Buffers: phosphate buffer, Na2HPO4-KH2POa; Tris buffer, Tris-HC1. Dehydrogenase assay. The dehydrogenase activity was measured by the rate of oxidation of NADH2 at 25 °. The decrease of ~s40 m# was recorded on a Photovalt Model 43 logarithmic recorder which was connected to the output of a Zeiss spectrophotometer. The reaction mixture contained 1.6.10 -4 M N A D H 2, 0.05 M Tris (pH 8.o), enzyme and 6 mM pyruvate for lactate dehydrogenase, the same plus 0.2 M NH4C1 for alanine dehydrogenase, or 0.006 M oxaloacetate for malate dehydrogenase. Since we found practically no NADH2-oxidase activity in our crude extract, the rate of N A D H 2 oxidation in the presence of pyruvate could be attributed to lactate dehydrogenase. The corresponding rate in the presence of pyruvate and NH4C1 should then be the sum of the individual rates of lactate dehydrogenase and alanine dehydrogenase. However, we have found, in partially purified lactate dehydrogenase free from alanine dehydrogenase, that lactate dehydrogenase activity was reduced by the NH4C1 to about 500/0 . The activity of alanine dehydrogenase in preparations containing lactate dehydrogenase was therefore approximately determined by the rate of oxidation with NH4C1 minus 0.5 × rate of oxidation without NH4C1. Dehydrogenase activities are defined as/,moles of N A D H 2 oxidized for one min under the above assay conditions. Estimation of protein. Protein was assayed by LowRY'S method o, with crystalline bovine serum albumin as standard, or in nucleic acid free preparations, by the absorbancy at 280 m#. EXPERIMENTS AND RESULTS

Isolation and crystallization of alanine dehydrogenase Growth and preparation of cells with high alanine dehydrogenase content. The nicotinic acid requiring strain was grown in the medium described above in 3oo-1 batches at 37 ° with strong aeration. When an inoculum of 2 • lO5 cells/ml was used, nicotinic acid became growth limiting after 16-18 h when the culture had reached a titer of about 5 "lOS cells/ml. After the cell growth had ceased, the bacteria were further aerated for 2 h, in order to maximize the derepression of alanine dehydroBiochim. Biophys. Acta, 92 (i964) 33-43

ALANINE DEHYDROGENASE OF e . subtilis

35

g e n a s e t These cells were harvested in a Sharples centrifuge and dried by acetone cooled at --20 ° . Preliminaryfractionation. This step is similar to that used by previous workers 2. IO g of acetone dried ceUs were suspended in 15o-2oo ml of 0.05 M Tris (pH 8.0) containing I mM mercaptoethanol, and digested with lysozyme (EC3.2.I.I7) (0.5 mg/ml) at 37 ° for 30 min. Subsequently nucleic acids were partially broken down b y 30 rain further incubation in the presence of ribonuclease (EC 2.7.7.16 ) (25 #g/ml), deoxyribonuclease (EC 3.1.4.5) (o.I #g/ml) and 2 mM MgCI2. The liquified extract was centrifuged at 30 ooo × g for 30 rain. After addition of MnSO 4 to the supernatant at a concentration of 0.05 M, a precipitate formed which was removed by centrifugation. All the subsequent procedures, including chromatography and dialysis, were carried out in the cold. Ammonium sulfate was added to the supernatant to give 90-95% saturation and, after standing for i h, the precipitate was collected by centrifugation. After removal of material which was soluble in 65% saturated ammonium sulfate, the insoluble fraction was extracted with 45% saturated ammonium sulfate. The extraction was repeated once more and the combined extract (about 12o ml) was reprecipitated with ammonium sulfate at 90-95% saturation. The precipitate was suspended in about 50 ml of o.oi M phosphate buffer (pH 7-7) and dialyzed against the same buffer. Calcium phosphate-gel column chromatography. The dialyzed extract (about 60 ml) was placed on a calcium phosphate-gel column (2 × 35 cm) buffered with o.oi M phosphate buffer (pH 6.8), and was eluted with phosphate buffer (pH 6.8) whose concentration gradually increased from o.oi M to 0.2 M. The elution gradient was produced by adding 0.05 M, o.I M and 0.2 M of phosphate buffer successively to a mixing chamber which originally contained 500 ml of o.oi M phosphate buffer. A typical elution pattern is shown in Fig. I. Alanine dehydrogenase could be separated from malate dehydrogenase and lactate dehydrogenase by this procedure,

2.2

l o o ~, 1.8

Q"

1.4

.5

60

o

~" '

1.o

80

! 40

~o.6

~ ~o

0.2 o

'

"

",

6 100 200 SO0 1460 500 6OO 7do Lr - - o ~ I M ~ o~3M' -JO.O3M~O75M .I.I IEffluent

;'

~

8O0 900 ~600 ~ 0 0 aoT~M ~2M "

"~ a

i

120( 0 LI

(ml)

Fig. I. E l u t i o n p a t t e r n f r o m a c a l c i u m p h o s p h a t e gel c o l u m n . T h e dialyzed 45-65 % a m m o n i u m sulfate e x t r a c t a b l e fraction (1.2 g p r o t e i n in 6o ml) was placed on a c o l u m n (2 × 35 cm) a n d e l u t e d w i t h p h o s p h a t e buffer of i n c r e a s i n g c o n c e n t r a t i o n . O - - O , a b s o r b a n c y ; © - - - G , a l a n i n e dehydrogenase activity; A ° --A, malate dehydrogenase activity; ×---×, lactate dehydrogenase activity.

Biochim. Biophys. Acta, 92 (1964) 33-43

36

A. YOSHIDA, E. FREESE

except that a weak malate dehydrogenase activity was detected in the alanine dehydrogenase peak. The bulk of the alanine dehydrogenase peak was precipitated with ammonium sulfate at 90-95% saturation, the precipitate was resuspended in about IO ml of o.oi M phosphate buffer (pH 7.7) containing I mM EDTA, and dialyzed against the same buffer. DEAE-Sephadex column chromatography and preliminary crystallization. The dialyzed solution obtained in the previous step was placed on a DEAE-Sephadex column (I × 30 cm) buffered with 0.02 M phosphate buffer (pH 7.7) containing I mM EDTA, and it was eluted with increasing concentrations of NaC1, from o.I M to 0.4 M. The gradient was produced by adding phosphate buffer containing 0.5 M NaC1 to a mixing chamber which contained 250 ml phosphate buffer containing o.I M NaC1. A typical elution pattern is shown in Fig. 2. This procedure removed the small amount of malate dehydrogenase contaminating the alanine dehydrogenase fraction of the previous step. The alanine dehydrogenase fraction was precipitated with ammonium sulfate at 90-95% saturation and redissolved in several ml of 0.05 M phosphate buffer (pH 7.7). Saturated ammonium sulfate solution was added until the suspension became slightly turbid. After cold storage overnight, a small precipitate 1.0

400

0.8

0

3 0 0 ~.

0.6

0.4

200 ?~

0.2

lOO & o

e o

Oo

I

i

0

50 100 NaCI 0.1M

150

200

300 250 P04 M Gradually

C3

350

E f f l u e n t .( ml )

Fig. 2. E l u t i o n p a t t e r n f r o m a D E A E - S e p h a d e x c o l u m n . P a r t i a l l y purified a l a n i n e d e h y d r o g e n a s e (peak f r o m c a l c i u m p h o s p h a t e c o l u m n , 14o m g protein) w a s placed on a D E A E - S e p h a d e x c o l u m n (i × 3 ° cm) a n d e l u t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n o f NaC1. Q---O, a b s o r b a n c y ; O- - -O, alanine dehydrogenase activity; A- " -A, malate dehydrogenase activity.

formed which was removed by centrifugation. Additional saturated ammonium sulfate solution was dropped into the supernatant until it became slightly turbid. When the suspension was kept overnight, a precipitate developed which showed a silky sheen. Small crystals could be seen in a microscope, but they were not free from amorphous precipitate. Further purification of alanine dehydrogenase by recrystaUization at this stage was not practical. Ecteola-cellulose column chromatography and crystallization. The crude crystals obtained in the previous step were dissolved in a few ml of o.oi M phosphate buffer Biochim. Biophys. Acta, 92 (1964) 33-43

ALANINE DEHYDROGENASE OF •.

37

subtilis

(pH 7.7) containing I mM EDTA, dialyzed against the same buffer and placed on an Ecteola-cellulose column (I × 30 cm) buffered with 0.02 M phosphate buffer (pH 7.7) containing I mM EDTA. The protein was eluted with gradually increasing NaC1 from o.I M to 0.3 M. A typical elution pattern is shown in Fig. 3.

0.3

150

0.2

100

C1 O

i '*o t c

0.1

\

~o N 8

\

<

0

50 NaCI

0.1M

I

I

100

150 • 0.3M

~0

I

200

Gradually

o

250 •

~

~ <

E f f l u e n t ( ml )

Fig. 3. E l u t i o n p a t t e r n f r o m Ecteola-cellulose c o l u m n . P a r t i a l l y purified a l a n i n e d e h y d r o g e n a s e (purified t h r o u g h D E A E - S e p h a d e x c o l u m n a n d fractional p r e c i p i t a t i o n w i t h a m m o n i u m sulfate, 25 m g protein) w a s placed on a n Ecteola-cellulose c o l u m n (I X 3 ° cm) a n d e l u t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n of NaC1. q ~ - O , a b s o r b a n c y ; O - - - O , a l a n i n e d e h y d r o g e n a s e a c t i v i t y .

The alanine dehydrogenase fraction was collected by precipitation with ammonium sulfate and redissolved in the smallest feasible amount of 0.05 M phosphate buffer (pH 7.7) containing I mM EDTA. After a small amount of insoluble material had been discarded b y centrifugation, saturated ammonium sulfate solution was added drop by drop until the solution became slightly turbid. Fine needle-like crystals developed after several hours (Fig. 4). Recrystallization could be performed without a significant loss in enzyme activity.

Fig. 4. P h a s e c o n t r a s t m i c r o g r a p h of a l a n i n e d e h y d r o g e n a s e crystals. Magnification 14oo.

Biochim. Biophys. Acta, 92 (1964) 33-43

38

A. YOSHIDA, E. FREESE SCHEME PURIFICATION

AND

CRYSTALLIZATION

I OF ALANINE

DEHYDROGENASE

Acetone dried cells (IO g) suspend in o.o 5 M Tris buffer (pH 8.o), digest with lysozyme, R N A a s e and DNAase at 37 °, centrifuge, 3 ° ooo × g for 3 ° min

Supernatant (approx. 17o ml) ] add MnSO 4 (final o.o 5 M), centrifuge, 3° ooo × g for 3° min

Supernatant (approx. 17 ° ml) I add (NH4) 2 SO 4 (6oo g/l), hold for I h, centrifuge, 3 ° ooo × g for 3 ° min

Precipitate e x t r a c t with 65 % satd. (NH4)2SO4, centrifuge

Precipitate e x t r a c t with 45 % satd. (NH4)zSO,, centrifuge, repeat 2 times

Extract (supernatant, total approx. 13 ° ml) add (NH,)2SO * (3oo g/l), hold for 3 ° rain, centrifuge

Precipitate suspend in o.oi M p h o s p h a t e buffer (pH 7.7), dialysis against p h o s p h a t e buffer

Dialyzed solution (approx. 6o ml) fractionation b y calcium phosphate-gel column c h r o m a t o g r a p h y

A lanine dehydrogenase fraction add (NH4)2SO 4 (6oo g/l), hold for 2 h, centrifuge

Precipitate dissolve in o.oi M p h o s p h a t e buffer (pH 7.7), dialysis against p h o s p h a t e buffer containing o.ooi M E D T A

Dialyzed solution I fractionation b y D E A E - S e p h a d e x column c h r o m a t o g r a p h y

Alanine dehydrogenase fraction ] a d d (NH~),SO, (6oo g/l), hold for 2 h, centrifuge

Precipitate dissolve in 0.o 5 M p h o s p h a t e buffer (pH 7-7), add satd. (NH4)2SO , until turbid, hold overnight, centrifuge

S~pernatant I add satd. (NH,),SO, until turbid, hold overnight, centrifuge

Precipitate dissolve in o.oi M p h o s p h a t e buffer (pH 7.7), dialysis against p h o s p h a t e buffer containing o.ooi M E D T A

Dialyzed solution ] fractionation b y Ecteola-cellulose column c h r o m a t o g r a p h y

A lanine dehydrogenase fraction ] add (NH4)2SO 4 (6o0 g/l), hold overnight, centrifuge

Precipitate [ dissolve in o.05 M p h o s p h a t e buffer (pH 7.7), centrifuge

Supernatant ] add satd. (NH,)~SO, until slightly turbid, hold overnight

Crystalline ala~ine dehydrogenase Biochim. Biophys. Acta, 92 (1964) 33-43

ALANINE DEHYDROGENASE

OF B .

subtitis

39

The various steps of purification are summarized in Scheme i. Yields and activities of alanine dehydrogenase, lactate dehydrogenase and malate dehydrogenase at different stages are shown in Table I. I t should be mentioned that all three dehydrogenases showed a greater total activity when they were more concentrated. All of the following experiments were carried out using alanine dehydrogenase recrystallized three times. TABLE

I

YIELD AND ACTIVITY OF DEHYDROGENASES IN FRACTIONATION

Total activity Step qf fvactionation

Crude extract Supernatant after addition of MnSO 4 Ppt. with (NH4)~SO 4 suspended in buffer (NH4)2SO4, 6 5 - 4 5 % f r a c t i o n Ppt. with (NH4)2SO 4 and suspend in buffer After dialysis

Total valine (ml)

A lanine dehydrogenase

Lactate dehydrogenase (xo 4 units)

'1o 8 - io a .IO ~ - IO a

1.5 1. 4 2.0 2. 4

0.95 i.o ---

o.51 o.49 0.50 0.46

4° 67

1.25" IO a 1.25 - lO 3

3.2 1.6

-o.84

o.72 o.69

14o

14o

0.76

o

0.o05

12 17o

-260

2.8 o

-o.48

o

15 230

-17o

__ o

0.79 o

-0. 4

--

__

__

0.63

31

1.36

o

o

--

2. 5

I7"

o.6

o

o

2. 5

--

1.9

1. 5

12"

1.6

17o 17o iio 126

T ota l ~rotein (rag)

4.0 2.95 2.2 1.3o

Malate dehydrogenase

C a l c i u m p h o s p h a t e - g e l - c o l u m n effluent Alanine dehydrogenase fraction Ppt. with (NH4)2SO 4 and dissolve in buffer Lactate dehydrogenase fraction Ppt. with (NH4)2SO 4 and dissolve in buffer Malate dehydrogenase fraction Ppt. with (NHa)2SO 4 and dissolve in buffer

DEAE-Sephadex-column

15

--

effluent

Alanine dehydrogenase fraction Ppt. with (NH~)~SO 4 and dissolve in buffer

48 2.0

Ecteola-cellulose-',olumn effluent Alanine dehydrogenase fraction Ppt. with (NH4)~SO 4 and dissolve in buffer Crystalline alanine dehydrogenase (3 t i m e s r e c r y s t . )

7°

* Protein was estimated from the absorbancy

a t 28o m # ~ / E Itern % = 6.4, s e e F i g . 6).

Measurement of sedimentation and molecular weight Sedimentation. Ultracentrifugation experiments were carried out in a Spinco Model E centrifuge by Dr. D. J. CUMMINGS. The protein was dissolved in o.I M phosphate buffer (pH 7.7) at concentrations of 1% and 0.3% for optical paths of 0.3 cm and I.O cm, respectively. Schlieren patterns of crystalline alanine dehydrogenase showed single and symmetrical sedimentation boundaries (Fig. 5), indicating a high degree of homogeneity of the protein. The sedimentation constant (s20,w) was calculated as 7.63 S at 1% and 8.45 S B i o c h i m . B i o p h y s . A c t a , 92 (1964) 3 3 - 4 3

4°

A. YOSHIDA, E. FREESE

Fig. 5. Schlieren pattern of crystalline alanine dehydrogenase in the centrifuge at 5° 74° rev./ min 32 rain after reaching final speed. The concentration of the protein was i % in o.i M phosphate buffer (pH 7.7). Optical path was o. 3 cm. at o.30/0 . The sedimentation constant at infinite dilution, obtained b y linear extrapolation of the two values was 8.8 S. Molecular weight. The molecular weight of the protein was determined b y the sedimentation equilibrium method 1°, using a Rayleigh interference optics in a Spinco Model E centrifuge. The interference patterns obtained b y Mr. L. GODFREY showed a high degree of homogeneity of the protein. The molecular weight was calculated as 228 ooo, employing a partial specific volume of 0.732 ml/g, which was calculated from the amino acid composition of the protein according to the m e t h o d described b y COHN AND EDSALL 11. The accuracy of the molecular weight determination b y this m e t h o d is estimated to be 4- 5 %.

Amino acid composition The crystalline enzyme was dialyzed against water and lyophilized. The weighed samples of dried protein were hydrolyzed for 20 h at lO8-11o ° in constant-boiling0.5 t i

0.4

i i t i

0.3

,"

c

' 0.2

',

', ', , ~

.a 0.1

0

L 240

260

280 300 Wavelength ( mJJ )

320

340

360

Fig. 6. Absorption diagram of crystalline alanine dehydrogeuase. - - - , 0.445 mg/ml protein in o.o5 M Tris buffer (pH 8.0) ; . . . . . , o.4o6 mg/ml protein in o.I N NaOH.

Biochim. Biophys. Acta, 92 (1964) 33-43

41

ALANINE DEHYDROGENASE OF B. subtilis

point hydrochloric acid in an evacuated sealed tube. The hydrolysates were evaporated to dryness in vacuo over NaOH. In order to estimate the cystine (or cysteine) content accurately, samples of oxidized protein were subjected to amino acid analysis. The procedure used for the oxidation of the protein with performic acid was essentially that described by SCHRAM, MOOR AND BIGWOOD1~. After oxidation, the reaction mixture was diluted with io vol. of cold water and the solution was immediately lyophilized. The samples were dissolved in a small amount of water and the solution was again lyophilized. The weighed samples of oxidized, dried protein were hydrolyzed as described above. Amino acid analysis was performed using a Phoenix automatic amino acid analyser. Tryptophan was estimated by measuring the extinction at 294. 4 and 280 m/z TABLE II AMINO ACID COMPOSITION OF B . subtilis ALANIN]~ DEHYDROGENASE Amino acid

C y s t e i c acid§ Aspartic acid M e t h i o n i n e sulfone Threonine§§ Serine§§§ G l u t a m i c acid Proline Glycine Alanine Cystine-SH§§t~ Valino Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophant Ammoniatt Total

% of amino acid non-oxidized~

oxidized

none lO.94 -4- o.18 none 7.84 4- o.03 3.90 ± 0.02 14.o i o.14 5.35 £ o.z2 7.18 4- o.18 11.2 -4- o.15 none lO.95 4- o.18 3.o9 4- 0.03 7.Ol 4-4-o.17 lO.52 q- o.12 5 .02 4- 0.05 2.09 ± o.03 7.79 4- 0.36 2.63 4- o.15 4.63 4- 0.09 0.48 4- 0.05 1.64 4- 0.04 116.66

0.64 lO.59 3.52 7.47 3.90 14.o6 5.55 7.22 lO.5O none lO.63 none 6.75 lO.44 4.54 1.99

% of amino acid residue**

-9.31 4- o.16 -6.5 ° ± o.16 3.23 ~ o.oi 12.31 ± 0.03 4.6o ± 0.o9 5.48 -4- o.o2 8.65 ± 0.28 o.42 9.13 4-4-o.14 2.72 4- o.o 3 5.94 4-4-o . i i 9.04 4- 0.04 4.52 i 0.05 1.84 ± 0.o 5 6.83 4- 0.33 2.33 4- o.13 4.17 ± o.08 0.43 4- 0.04

Calculated***molar rat io

-42.9 ± -34.1 419.7 ~5o.6 ± 25.1 i 50.8 464.7 -42.o 48.8 4i i . o 427.8 442.3 414. 7 ± 6. 7 428. 3 49.0 414.o 41.2 4-

0.7 o. 7 o.i o.i 0.4 0.2 2.0 0.7 o.I o. 5 o.2 o.2 0.2 1. 3 0. 5 o. 3 o.i

A ssumed**** molar" ratio

-43 -34 20 51 25 51 65 2 49 ii 28 42 15 7 28 9 14 i

* M e a n v a l u e a n d d e v i a t i o n s of c o m p l e t e i n d e p e n d e n t d u p l i c a t e a n a l y s i s . ** M e a n v a l u e a n d d e v i a t i o n s of a n a l y s i s of n a t i v e a n d p e r f o r m i c acid o x i d i z e d p r o t e i n . The c o n t e n t s of m e t h i o n i n e sulfone a n d t y r o s i n e of p e r f o r m i c a c i d o x i d i z e d p r o t e i n h a v e n o t been i n c l u d e d in t h e m e a n value, b e c a u s e of u n c e r t a i n t y of t h e i r re c ove ry. *** M o l a r r a t i o of i n d i v i d u a l a m i n o a c i d residues c a l c u l a t e d a s s u m i n g t h a t c y s t i n e r e s i d u e is I.OO. . . . . T h e n e a r e s t i n t e g r a l n u m b e r to t h e c a l c u l a t e d m o l a r ra t i o. § The r e c o v e r y of c y s t e i c acid in p e r f o r m i c acid o x i d a t i o n is a s s u m e d to be 90 % (see rcfs. 12,

13). §9 T h e r e c o v e r y a f t e r h y d r o l y s i s is a s s u m e d to be 9 0 % . H§ The r e c o v e r y a f t e r h y d r o l y s i s is a s s u m e d to be 8 0 % . §t§§ C a l c u l a t e d f r o m t h e c y s t e i c acid c o n t e n t . t E s t i m a t e d f r o m t h e e x t i n c t i o n of t h e p r o t e i n (see t e x t ) . t t N o t i n c l u d e d in t h e t o t a l . B i o c h i m . B i o p h y s . A c t a , 92 (1964) 33-43

42

A. YOSHIDA, E. FREESE

on o.I N N a O H solution correcting for spurious absorption as described by GOODWlN (Fig. 6). The tyrosine content estimated from the extinction was 4.9 ° 4- 0.05%, which agrees with the value obtained by amino acid analyser, i.e., 5.02 4- 0.05%. The amino acid composition of alanine dehydrogenase is presented in Table II. Molar ratios of amino acid residues, taking that of cystine as I.OO, are as follows: Asp, 43; Thr, 34; Ser, 20; Glu, 51; Pro, 25; Gly, 51; Ala, 65; CySH, 2; Val, 49; Met, I I ; iLeu, 28; Leu, 42; Tyr, 15; Phe, 7; Try, I; Lys, 28; His, 9; Arg, 14 .

AND MORTON 14

DISCUSSION

Throughout purification, the specific activity of the three dehydrogenases, alanine dehydrogenase, lactate dehydrogenase, and malate dehydrogenase, was always higher in concentrated than in dilute solutions. An increase in the total activity of alanine dehydrogenase during purification from B . cereus has also been observed by McCoRMICK AND HALVORSON 4. They attributed this effect to the removal of N A D H 2 oxidase which contaminated the crude extract. In our case this explanation is not acceptable since the crude centrifuged extract already did not contain any significant NADH~-oxidase activity. Moreover, even crystalline alanine dehydrogenase exhibited a greater specific activity when dissolved at high concentration ( > I.O mg/ml) and assayed immediately after dilution, than when it was kept at low concentrations ( < o.I mg/ml) for more than 5 min before it was assayed. These observations as well as light scattering data, which will be published elsewhere, show that alanine dehydrogenase decomposes on dilution into smaller units with lower specific activity. I f one assumes the shape of the protein as a prolate spheroid one can calculate its major dimensions from the molecular weight 228 ooo, the partial specific volume 0.732 ml/g, and the sedimentation constant 8.8 S at infinite dilution. The major axes are then 35 and 4.0 m/~, respectively. The amino acid analysis presented unusual difficulties since tryptophan and cystine, which are most difficult to estimate quantitatively, were also the two least frequent amino acids. No trace of cystine was detected in the hydrolysates of nonoxidized protein. The presence of cystine or cysteine in alanine dehydrogenase was established only by the appearance of cysteic acid after oxidation of the protein. This complete loss of cystine during hydrolysis is especially unusual since the protein contains no appreciable amounts of carbohydrate and only little tryptophan. The molar ratios of amino acid residues shown in Table U were based on the cystine (or cysteine) content. For this purpose it was assumed that lO% cystine (or cysteine) were lost in the performic acid oxidation procedure. The actual loss m a y be as low as 5 %, since after oxidation of protein the recovery of cysteic acid usually ranges between 9 ° and 95% (refs. 12, 13). The amino acid composition can be used to estimate the possible size of a subunit of alanine dehydrogenase. I f the enzyme contains only identical subunits and if each subunit has one cystine, or two cysteine, residues, its molecular weight would be 53 ooo to 56 ooo and the enzyme would contain, in its concentrated form, four such subunits. This size of a subunit seems most likely if one assumes that the calculated molar ratio of 1.2 to 1.3 tryptophan residues is closest to I. However, the determination of tryptophan by means of ultraviolet absorbance is not too accurate, especially Biochim. Biophys. Acta, 92 0964) 33-43

ALANINE DEHYDROGENASE OF B. subtilis

43

in a protein of low tryptophan content. We can therefore not exclude the possibility that each subunit contains one cysteine and one tryptophan residue and has a molecular weight of about 27 ooo. ACKNOWLEDGEMENTS

The authors are indebted to Dr. D. J. CUMMINGS for the sedimentation measurements and to Mr. L. GODFREY for the determination of molecular weight.

REFERENCES i j . M. WIAME AND A. PII~RARD, Nature, 176 (1955) lO73. 2 A. PII~RARD AND J. M. WIAME, Biochim. Biophys. Acta, 37 (196o) 490. s R. J. O'CONNOR, Thesis for the degree of Doctor of Philosophy, Univ. Microfilms, Mic 61-1546, University of Wisconsin (1961). 4 N. G. McCoRMICK AND H. O. HALVORSON, J. Bacteriol., 87 (1964) 68. 5 E. FREESE AND J. OOSTERWYK, Biochemistry, 2 (1963) 1212. 6 E. FREESE, Biochim. Biophys. Acta, 81 (1964) 442. A. TISELIUS, S. HJERT~N AND D. LEVlN, Arch. Biochem. Biophys., 65 (1956) 132. s C . ANAGNOSTOPOI:LOS AND J. SPIZlZEN, J. Bacteriol., 81 (1961) 741. 9 0 . H. LOWRY, N. J. ROSENBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. x0 D. A. YPHANTIS, J. Biochemistry, 3 (1964) 297. 11 E. J. COl'IN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New York, 1943, p. 37 o. 12 E. SCHRAM, S. MOOR AND E. J. BIGWOOD, Biochem. J., 57 (1954) 33. 13 p. E. WILCOX, E. COHEN AND W. TAN, J. Biol. Chem., 228 (1957) 999. x4 T. W. GOODWlN AND R. A. MORTON, Biochem. J., 4 ° (1946) 628.

Biochim. Biophys. Acta, 92 (1964) 33-43