Eur. J. Biochem. 83, 307-311 (1978)

Purification and Characterization of Alanine Carrier Isolated from H-Proteins of Bacillus subtilis lwao KUSAKA and Keiko KANA1

Institute of Applied Microbiology, University of Tokyo (Received July 29/0ctober 19, 1977)

Alanine transport carrier was isolated and purified from H-proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 pM. The binding was inhibited by p-chloromercuribenzoate and the inhibition was reversed by dithiothreitol.

In a previous paper [1] we have reported that hydrophobic proteins (H-proteins), which accumulate in membrane fractions of Bacillus subtilis, contained several intrinsic membrane proteins including amino acid carrier [2], H-proteins were insoluble in usual buffers but were found to be soluble in 8 M urea solution. Therefore, several membrane proteins could be purified directly from H-proteins without using detergents. This report concerns isolation, purification and characterization of alanine carrier from H-proteins of Bacillus suhtilis. MATERIALS AND METHODS Preparation q/' H-protein H-protein of Bacillus subtilis was isolated as previously described [2] and dissolved in 50 mM Tris-HC1 buffer (pH 7.4) containing 8 M urea. The solution was stood for about 1 h at 0 "C and then centrifuged at 140000 x g for 2 h. After centrifugation, the supernatant was used for the starting material (solubilized H-protein). Gel Electrophoresis Preparative polyacrylamide gel electrophoresis was performed using an apparatus for continuous gel electrophoresis type TB-2 (Mitsumi Kagaku Co., Tokyo). Urea/polyacrylamide gels used for purification of carrier or disc gel electrophoresis were the same as described by Jovin et al. [3]. For purification of the _

_

~

Abhreuzations. ClHgBzOH, p-chloromercuribenzoate.

carrier, a cylindrical 150-ml gel was used. Electrophoresis on disc gel of polyacrylamide containing dodecyl sulfate was performed as described by Weber and Osborn 141. Disc gels were stained with 0.025% Coomassie brilliant blue in 45% methanol and 10% acetic acid for 1.5 h and destained in 7.5 % acetic acid and 5 % methanol. Assay of Alunine Carrier Activity For assay of alanine carrier activity, samples containing carrier protein were constituted into vesicles with lipid from Bacillus suhtilis (consists mainly of phosphatidyl glycerol, 60 %, and phosphatidyl ethanolamine, 25 %) and the transport energy was supplied as a membrane potential introduced by K' diffusion via valinomycin 151. Lipids (5 mg) were suspended in 1 ml 50 mM Tris-HC1 buffer (pH 7.4) containing 2% sodium cholate, 1% sodium deoxycholate (lipid/detergent mixture), and the sample containing alanine carrier (20 - 50 pg protein) was added to the lipid/detergent mixture. Then the mixture was dialysed against 50 mM Tris-HC1 buffer (pH 7.4) for 15 h at 4 "C. The dialysate (vesicles) was suspended in 0.5 M potassium phosphate buffer (pH 8.0) and incubated for 30 min at 50 "C (process for potassium loading) and transfered to an ice bath. Then MgCI, was added to a final concentration of 20 mM and centrifuged at 140000 x g for 30 min. The precipitate was washed with 0.4 M sucrose/ 20 mM MgCl, and suspended in 0.4 M sucrose/20 mM MgCl, . The K+-loaded vesicles were suspended in 0.5 ml of solution containing final concentration of 50 mM Tris-HC1 (pH 7.4), 20 mM MgCI,, 0.4 M sucrose, and 10 pM ~-[U-'~C)alanine (1 pCi/ml). After

308

Purification of Alaninc Carrier

incubation for 3 min at 27 "C, 1 pg valinomycin in 1 pl ethanol was added. At intervals, 50-pl samples were filtered through membrane filters (Sartorius, 0.45 pm pore size) and washed with 0.4 M sucrose/20 mM MgCI,. The filters were dried and their radioactivities were assayed in a gas-flow counter. Initial rate of transport was estimated and the activity was shown as nmol alanine transported min-' mg protein ' ~

As.srij.,fi)rB i d i n g of Alminr to the Carrier

Binding of' alanine to the carrier was measured by the gel filtration method described by Hummel and Dreyer [6]. A Sephadex G-15 column (0.4 x 15 cm) was equilibrated with 50 mM buffer containing 1.5 pM ~-[U - ' ~ C ] a l an i ne (0.25 pCi/ml) and 0,s mM dithiothreitol. Carrier protein (5 - 10 pg) was incubated in 25 p1 of the solution at 25 "C for 20 min and then applied on the column. Elution was carried out with the samc solution at a flow rate of 0.1 m1/2 min and 0 , l -ml fractions were collected,

c'action n m c e :

Fig. 1. Prepurutiw e1eclrophoresi.c of H-protcirr 0ii p o / ~ i i ' . r ~ / c r r i i i ~ f i ~ gels (1004) contuining 8 A4 urru. Formation o f urea polyacrylamide gels and the electrophoresis were performed a s described by Jovin et ul. [ 3 ] . About SO mg H-protein after (h'H4),SO4 fractionation was applied on a gel and electrophoresis was performed at a constant current of 75 mA. 2-1111 fractions were collected. An arrow indicates the position the tracking dye (bromphenol bluc) uas eluted. The bar shows fractions containing alanine carrier

Pl.Otc~r11E\! /r?lc/tIOll

Protein\ mere estimated by the method described by Lowry c( ( i l [71

Table 1. Stimrnury of purificarion ofulanb7c czirrier. Purification step

Total protein

Total activity

m&

nmo1,min

Recovery Specific act ivil y nmol min mg- I

RESULTS H-protein was dissolved in 50 mM Tris-HCI buffer (pH 7.4) containing 8 M urea and fractionated by (NH,),SO,. The carrier was recovered in 3 S - 5 5 ' x saturation and the specific activity was increased about 5-fold by the fractionation, The precipitate of 35 - 55 "/, (N€34)2S04saturation was dissolved in SO mM TrisHCI bufl'er IpH 7.4) and dialysed overnight against the same bufl'er, The dialysate was again dissolved in 8 M urea solution and the solution was applied on a polyacrylamide gel (10';;;) containing 8 M urea. Electrophoresis was performed at a constant current of 75 mA, Alanine carrier n.as the first peak eluted after the tracking dye (bromphenol blue) was eluted (Fig. 1). The results o f a typical purification of alanine carrier are summarized in Table 1 , Specific activity was increased about 300-fold during the purification. Fractions 9 -- 12 of preparative electrophoresis (Fig 1) were collected and solid (NH,),SO, was added until the carrier was salting out (about 65% saturation). The precipitate was collected by centrifugation and dialysed against 50 mM Tris-HCI buffer (pH 7.4), The carrier protein was hydrophobic and insoluble in usual buffers, Thcrclhre. the protein suspension was homogeni;.ed by ;I Teflon homogenizer for use in the experiment,

~

Solubilized H-protein 380 3 5 - 5 5 % (NH,),S04 saturation 48 Fractions 9 12 of 0 67 urea gel electrophoresis

60.8

I 00

0.16

37.4

61

0 78

27.3

14

'

~

40.7

Polyacrylamide disc gel electrophoresis in the presence ofurea or dodecyl sulcate was used to examine the purity of the carrier protein. As shown in Fig, 2 and 3, the purified carrier showed a single band in gel electrophoresis, For determination o f molecular weight by dodecyl sulfate gels, it is important to show that the carrier protein moves normally in the gels, For this purpose, electrophoresis on gels with different concentrations of acrylamide was per.formed. As shown in Fig. 4. the carrier moved normally in the gels (5-120,, gel concentrations). The molecular weight of the carrier was estimated to be about 7500 by the gel electrophoresis (lo?( gel) as shown in Fig. 3, A Lineweaver-Burk plot of alanine transport activity of the vesicles formed by the purified carrier is shown in Fig. 5. The data indicated a A,, ofabout 3 pM.

309

1. Kusaka and K. Kanai

A

6

0

2

I

3

l/bj (KM '1 Fig. 5. Lineweaver-Burk plot of alanine transport activities of the aesicles formed by purijied carrier Fig. 3

Fig. 2

Fig. 2. Urea polyacrylamide disc gel electrophoresis of solubilized H-protein cind purijied alanine carrier. 10 pg alanine carrier (A) and 75 pg H-protein (B) were applied on the gel. Arrows indicate alanine carrier Fig. 3. Sodium dodecyl sulfate polyucrylamide disc gel electrophoresis ofpurijied alanine carrier. Gel electrophoresis in 10% acrylamide, 0.25% bisacrylamide gels containing 0.1 dodecyl sulfate was performed as described by Weber and Osborn [4]. 10 pg purified alanine carrier was applied on the gel

10

F

5-

0

1 .-

\*

05-

01

0

5 10 Gei concentration ('lo)

Additions

None Carbonylcyanide m-chlorphenylhydrazone NaCN Dicyclohexylcarbodiimide ClHgBzOH

iL

m

Table 2. Effect of aarious inhibitors on alunine transport Vesicles formed by purified carrier protein were loaded with potassium phosphate as described in the text. The reaction mixture (0.5 ml) contained final concentrations of 50 mM Tris-HC1 buffer (pH 7.4) 0.02 mM MgCI,, 0.4 M sucrose and 10 pM ~-[U-'~C]alanine. After incubation for 3 min at 27 "C with the inhibitors listed, valinomycin (1 pg) was added. Samples were filtered and washed and their radioactivities were counted

15

Fig. 4. Ferguson plot of alanine carrier. Electrophoresis of alanine carrier on dodecyl sulfate/polyacrylamide gels with different concentrations of acrylamide was performed as described by Hedrick and Smith [lo]. Bisacry1amide:acrylamide ratio was fixed at 1 :40

The effect of various inhibitors on alanine uptake of the vesicles formed by the purified carrier was next studied. As shown in Table 2, uptake activity of the vesicles, which was dependent on an artificial membrane potential, was blocked by carbonylcyanide m-

Concentration of inhibitor

Activity

Relative activity

mM

nmol min-' mg-'

%

40.72

100

0.02 2

0.00 37.53

0 92.2

0.1 0.2 0.05 1

43.55 2.30 6.14 10.26

107.0 5.6 15.1 25.2

chlorophenylhydrazone (0.02 mM), but not by dicyclohexylcarbodiimide (0.1 mM). The uptake activity was also inhibited by 0,05 mM p-chloromercuribenzoate (ClHgBzOH) and by 1 mM N-ethylmaleimide. Effects of temperature on the uptake of alanine are shown in Table 3 . There was almost no uptake at 10 "C, and the optimum temperature was 25 "C, The rate decreased at higher temperatures. The binding of alanine to the carrier was examined by the gel filtration method as described by Hummel and Dreyer [6]. A Sephadex G-15 column equilibrated with [14C]alanine was used for this purpose. One of the examples of the elution profile of ['4C]alanine accompanying the passage of alanine carrier through the column is shown in Fig. 6. A complex formed by

310

Purification of Alanine Carrier

Relative activity

Table 4. Effecr of p-cliloronicrcuriben~ou/emil dithiothwitol on binding aJ' alanine / o the carrier Binding of ['4C]alanine was determined as described in the text. Binding was tested in 50 mM Tris-HC1 buffer (pH 7.2). The control comprised carrier and buffer containing [14C]alanine only

f.,'

Conditions

Table 3 Effect of tmiprrmture on alanine transport Reaction mixture\ and experimental procedures were the same as in Table 2 Temperature

Activity nmol min

C 10 15

mg-

0 14 9.30 38.30 34.52 28.50

2s 30 37

0.3 24 100 90 74

L dc'

or

20

concn mM

Control Dithiothreitol ClHgBzOH ClHgBzOH + dithiothreitol

pmol/pg protein 98 138 8.5

0.5 0.05 0.05 0.5

68.5

25

&be.

Fig 6 Binding of [14C]olatiine to the purified cart ier with a Sephadex G-15 column c~yuilihraied itz/h /he anzino m i d A Sephadex G-15 column (0 4 x 15 Lm) nlns equilibrated with 50 mM Tris-HC1 buffer (pH 7 2) contdining 1 5 pM ~ - [ ' ~ C ] a l a n i n e(0 25 kCi/ml) and 0 5 mM dithiothreitol 2 3 pgcdrrier wasincubated with ['4C]alanine 5olution for 20 min dt 25 C prior to application on the gel Other details are presented in the text

in

)

""Yll"

addition

_ ,

15

d

I

Alanine

L'%lanine oolind (nrnol)

Fig. I . Scatchard plot of binding of alanine to the carrier. Binding assays were performed with 2.3 pg carrier and varying amounts of alanine

\ of alanine was determined as Fig.6. The buffers (50 mM) used were Tris-HCI (pH 8.0, 7.6 and 7.2), Tris-maleate (pH 6.7 and 6.2) and sodium acetate (pH 5.5 and 4.6)

volume. The binding of alanine as a function of substrate concentration was examined. and the data, plotted according to the method of Scatchard [Sl, indicated a Kd of about 0.2 pM and that 1 mol alanine was bound/mol carrier (Fig. 7 ) . Gordon et al. [9] had shown that the prolinebinding activity of partially purified carrier was inhibited by ClHgBzOH and the inhibition was reversed by dithiothreitol. Therefore, we have examined the effect of ClHgBzOH and dithiothreitol on the binding. As shown in Table 4, the binding activity was inhibited by 0.05 mM ClHgBzOH and the inhibition was reversed by 0.5 mM dithiothreitol These results indicate that -SH groups may be involved in the binding, The effect of pH on the binding was studied and is shown in Fig. 8. No binding was observed at acid or alkaline pH and the pH-optimum for the binding was at around pH 7.0.

I. Kusaka and K. Kanai

DISCUSSION H-proteins, accumulated in the membrane fraction of Bacillus subtilis, contain various membrane proteins [2]. The proteins are free from lipids and easily dissolve in 8 M urea solution, therefore several membrane protein components can be isolated and purified without using detergents. Alanine carrier was the first one which we tried to isolate and purify from H-proteins. The carrier was stable in 8 M urea solution and was purified as presented in this paper. The purified carrier was homogeneous as judged by polyacrylamide disc gel electrophoresis and the minimum molecular weight was around 7500. Binding of alanine to the carrier was tested by an equilibrated column of Sephadex G-15 and alanine was bound only at neutral p H (pH 6.5-7.5): At pH 7.2 the carrier formed a complex with alanine in a molar ratio of 1 :l. The dissociation constant of the binding was 0.2 pM and the value was about 1/15 of K,,, of alanine uptake. The binding may be dependent on an - SH group and was inhibited by p-chloromercuribenzoate. The inhibition was reversed by dithiothreitol. The uptake activity was temperature dependent and almost no activity was found at 10 "C. This may be reflected by the lipid phase in the vesicles and 10 "C may be below the transition temperature of the lipid

311

used in the experiment. The carrier may not be mobile or valinomycin may not work at the temperature. Purified alanine carrier also had a affinity for glycine and the vesicles formed by the purified carrier could accumulate glycine in the vesicles. The transport activities for other amino acids were tested also by the vesicles formed by the purified alanine carrier and L-glutamate, L-leucine, L-valine, L-methionine, L-proline, L-threonine, L-serine, L-lysine and L-aspartate were not accumulated by the vesicles,

REFERENCES 1. Kusaka, I. (1974) Biochim. Biophys. Acta, 345, 62 - 73. 2. Kusaka, I., Hayakawa, K., Kanai, K. & Fukui, S. (1976) Eur. J . Biochem. 71,453 -458. 3. Jovin, T., Chrambach, A. & Nanghton, M. A. (1964) Anal. Biochem. 9, 351 - 369. 4. Waber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 44064412. 5. Hirata, H., Altendolf, K. & Harold, F. M. (1974) J Biol. Chem. 294, 2939 - 2945. 6. Hummel, J . P. & Dreyer, W. J. (1962) Biochirn. Biophys. Acta, 63, 530- 532. 7. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275. 8. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 5, 660-672. 9. Gordon, A. S., Lombardi, F. T. & Kaback, H. R. (1972) Proc. Natl Acad. Sci. U.S.A. 69, 358 - 362. 10. Hedrick, J. L. & Smith, A. J. (1968) Arch Bzochem. Biophys. 126, 155-164.

I. Kusaka and K. Kanai, Institute of Applied Microbiology, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, Japan 113