THE JOURNAL OF BIOLOGICAL. CHEMISTRY

Printed in U.S.A. Vol. 257, No. 24, leaue of December 25, pp. 15167-15173,1982

Purification and Properties of the Biotin Repressor A BIFUNCTIONAL PROTEIN*

(Received for publication, June 17, 1982)

Max A. EisenbergS, Om Prakashg, and Shu-Chi Hsiung From the +Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York 10032 and the 6Denartment of Molecular Virolom, Sloan Kettering Institute for Cancer Research, ”

New York, New York 10021 .

Definitive evidence is presented for the bifunctional nature of the biotin repressor protein which possesses both regulatory and enzymatic activities. The repressor protein can activate biotin in the presence of ATP to form biotinyl-5’-adenylate, the co-repressor which re- mains tightly bound to the repressor protein. This com- plex can either bind to the operator site and inhibit transcription or transfer the biotinyl moiety to a lysine residue of the apoenzyme of acetyl-coA carboxylase. The two activities were coincident throughout a puri- fication procedure which resulted in a 3500-fold in- crease in activity. Gel electrophoresis of the purified preparation, under native or denaturing conditions, showed three proteins with the activity corresponding to the major protein band of apparent M, = 34,000. On gel exclusion chromatography, the activity was also associated with a protein of M, varying from 37,000- 44,000, indicating the protein is monomeric. The occa- sional appearance of multiple bands with biological activity in the native gels suggests that the repressor protein can also exist in multimeric forms. On chro- matofocusing, the repressor activity and the holoen- zyme synthetase activity were coincidental, with the peak of activity at pH 7.2, the isoelectric point. Only a single protein band with M, = 34,000 was observed on SDS gel electrophoresis of all fractions showing activity.

Regulatory mutants of the biotin operon, bioR, have been isolated by Eisenberg (1) on the basis of their resistance to the biotin analogue, a-dehydrobiotin, a growth inhibitor. The enzymes of the biotin operon are fully derepressed in these mutants, which also exhibit high biotin excretion. Similar mutants have also been isolated by Pai (2) on the basis of the latter property. Co-transductional analysis by both groups has placed bioR at min 89, near the bfe locus, on the Escherichia coli genetic map. A rifampicin-dominant transducing phage, Xdrifd isolated by Kirschbaum and Konrad (3), has been found by transductional analysis to carry the bioR gene and has been used to amplify the &OR gene product, the biotin re- pressor protein. With an in vitro protein-synthesizing system, Prakash and Eisenberg (4) have been able to demonstrate the repression of the synthesis of two of the biotin biosynthetic enzymes when the system was fortified with both the partially purified repressor protein and biotin. In a subsequent study of

* This investigation was supported by Public Health Service Grant AM-14450 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

the interaction of the repressor protein-biotin complex with the operator site of the biotin operon, these investigators concluded that the true co-repressor is biotinyL5’-adenylate. The reason that biotin could function in the in vitro system was because the partially purified repressor protein prepara- tion could also activate biotin in the presence of ATP to form biotinyl-5’-adenylate (5). No definitive conclusion could be reached whether the repressor activity and the enzymatic activity are properties of a single protein.

A bir mutant, isolated by Campbell et al. (6) , appears similar to the bioR mutants in that the enzymes of the biotin operon are derepressed. This mutant, in contrast, requires high biotin concentrations for growth and shows decreased permeability to biotin. The suggestion was made that a single protein may be responsible for this pleiotropic phenotype. Although initial mapping placed the mutation near thi, a subsequent analysis by Pai and Yau (7) indicated that bir and bioR probably map at the same locus. In an extensive bio- chemical and genetic analysis, Barker and co-workers (8-10) have determined that bir is the structural gene for the enzyme, biotin holoenzyme synthetase. This enzyme activates biotin in the presence of ATP and links it covalently to a lysine moiety of acetyl-coA carboxylase, the predominant biotin enzyme of E. coli. Complementation analysis disclosed the complete or nearly complete overlap of the bir and birR genes, suggesting their possible identity. In addition, these investi- gators have been able to demonstrate that a partially purified preparation of the holoenzyme synthetase protein would pro- tect the operator site from the TaqI restriction enzyme, but only in the presence of biotin-5”adenylate. Thus, the evidence from both laboratories suggests a bifunctional protein with both regulatory and enzymatic properties.

The present study reports the isolation of a homogeneous repressor protein. We have demonstrated that a single protein possesses both regulatory and enzymatic activities: a unique property among repressor proteins thus far isolated.

MATERIALS AND METHODS‘

RESULTS

A summary of the purification procedure, shown in Table I, indicates about a 3500-fold purification of the repressor protein. This actually represents a 24,000-30,000 increase in

’ Portions of this paper (including “Materials and Methods” and Figs. 1-6) are presented in miniprint at the end of this paper. Mini- print is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chem- istry, 9650 RockviUe Pike, Bethesda, MD 20814. Request Document No. 82M-1623, cite authors, and include a check or money order for $4.40 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

15167

15168 Purification and Properties of the Biotin Repressor

TABLE I

DUE"ll"1Q. I Pll 7.b sm 10.5 115 10 .5 l l .b 60 3 . 5 I 35b 17.4 5.4

m p h o c . l l u l a * . l b 1 1 . 0 3030 145 7 . 9 3e 48

I l l - e . l l u l o r I 9.6 4.1 1303 1015 1.0 38 b75

4970 1 3 7 74

D U I " l l Y l 0 " 11

3516 5317 1-5 D m - l l u l o r I1 13 0.W 1503 1307 4.b 11 17b9

e m 1.)) 610

p 6 . 5 . t u b . a l b 3 . 9 0.31 le91 11.117 1.5 7 3705 3176 a 7 4 l e 3 b

*P.LC.- 8 p C l K l c *CtlVIty.pmol. OK b lo t ln bod/- pr0t.h.

IP.AC.- l p r l r l c .ct~ric)r. pmoh OK blocln ineorporat .d/mm/q prot.~n.

PH

FIG. 7. Preparative isoelectric focusing. The procedure is de- scribed under "Materials and Methods." A DNA-cellulose I1 prepa- ration with a biotin-binding activity of 3968 pmol/ml was used. Biotin binding activity (O), repressor activity (A), [:"P]DNA filter-binding activity (A).

biotin-binding activity over the crude extract prepared from the parental strain, since the activity increases 8-10 fold on induction of the rip18 lysogen. The holoenzyme synthetase activity is also included in the table and it can be seen to parallel the purification observed with the biotin-binding as- say.

Properties of the Purified Repressor Protein-The most purified repressor preparation was stable over a 6-month period at -20 "C, but rapidly lost activity on repeated freezing and thawing. The PI of the protein was determined to be 7.2 by preparative isoelectric focusing. The activity profile shown in Fig. 7 indicates that the three activities, biotin binding, in vitro repression, and r2P]DNA binding, are coincidental.

The peak fraction of the DEAE-cellulose I1 chromatogram gave three protein bands on polyacrylamide gel electropho- resis as indicated in Fig. 8, a and b. When duplicate electro- phoretic gels were sliced and assayed for biotin binding and biotin activation activities, identical RF values were obtained for both which corresponded most closely to the RF value of the most intensely stained protein band. A more accurate

a b C

FIG. 8. Native disc gel electrophoresis of peak fractions from DEAE-cellulose I1 chromatography, pH 6.5. Acrylamide concen- tration was 5.68 for the running gel. Lane a, fraction 16; lanes b and c, fraction 17. Protein applied was 7 and 8 pg, respectively. Gels b and c were run on consecutive days.

comparison between the measured mobility of the stained gel and the activity was made with a vertical slab gel. In Fig. 9, the stained gel was drawn to the same scale as the biotin activation activity and only two bands are apparent. It is evident that the biotin activation activity coincided with the major protein band. On occasion, we have also observed six to seven bands with the same preparation as indicated in Fig. 8c. This gel was run 1 day later than the gel shown in Fig. 8b. When the biotin activation activity was determined in a companion gel, a number of activity peaks were observed. The RF values corresponded to many of the stained bands, indi- cating that they represented multimeric forms of the repressor protein.

Molecular Weight-Gel exclusion chromatography carried out over a wide range of pH and sodium chloride concentra- tions gave M, values ranging from 37,000-44,000. The samples analyzed were from the 60% ammonium sulfate and the DNA- cellulose chromatography steps. A typical analysis in which a value of 42,000 daltons was obtained is shown in Fig. 10. On one occasion, a component of M, - 90,000 was observed in the DNA-cellulose preparation but this component comprised less than 1% of the total activity. SDS'-polyacrylamide electro- phoresis (Fig. 11) shows that the DEAE-cellulose I1 chroma-

* The abbreviation used is: SDS, sodium dodecyl sulfate.

Purification and Properties of the Biotin Repressor 15169

0

N 6 $ x

E 4

2

10 20 30 mm

90

FIG. 9. Polyacrylamide slab gel electrophoresis and the bio- tin activation profile of fraction 16. Protein samples, 7 and 28 pg, were placed in adjacent wells of a 7% polyacrylamide slab gel. On completion of the run, the gel was cut lengthwise to remove the entire area under the well containing the higher protein concentration. This was sliced into 1.15-mm sections which were assayed for biotin acti- vation activity. The remaining gel was stained with Coomassie blue.

I 1.4 1 .a 2.2

Ve I VO

FIG. 10. Molecular weight determination by gel exclusion chromatography. One ml of the 60% ammonium sulfate fraction (biotin-binding activity of 302 pmol/ml) was chromatographed on a Sephacryl S-200 column (1.6 X 92 cm). The elution buffer was 0.01 M sodium phosphate, pH 6.5, containing 0.5 M NaCI, 5% glycerol, 1 X 10 M dithiothreitol. The biotin binding activity and Am, were determined on each fraction. The column was previously equilibrated with the following molecular weight (M. W.) protein markers: 1, bovine serum albumin, 66,200, 2, ovalbumin, 45,000, 3, chymotrypsinogen, 25,700. The arroul indicates a VJV, of 1.78 for the repressor protein with M , = 42,000.

-92.5 K -6 6.2

-4 5

a - -3 1

-21.5 -1 4.4

tography reduced the number of protein bands to three, corresponding to M, - 98,000, 26,000, and 34,000 in the order of increasing staining intensity.

While the high molecular weight component could be ex- cluded on the basis of the results from gel exclusion chroma- tography, it was essential to determine which of the two remaining proteins was associated with the biological activity. Two-dimensional electrophoresis was therefore carried out with the most purified fraction. The native gel was sliced, and each slice was prepared for SDS electrophoresis according to the method of O’Farrell (19) and placed into a separate well of an SDS gel. After staining the gel, only the M , = 98,000 and 34,000 proteins were visible. The migration of the latter pro- tein appeared to be coincident with the distribution curve for the biotin-binding activity obtained on a companion native gel (data not shown).

Chromatofocusing-Although the two-dimensional electro- phoresis strongly suggested that the biological activity was associated with the M , = 34,000 protein, it was essential to obtain a more homogeneous protein preparation to determine if both activities resided in the same protein. Chromatofocus- ing over the pH range of 6.0-8.0 proved to be most successful. In Fig. 12 are shown the results of a chromatofocusing chro- matogram on another repressor preparation with somewhat lower specific activity. This preparation had also been purified through the DEAE-cellulose I1 stage but the first DEAE- cellulose step was omitted. It is apparent that biotin binding, in vitro repression, and holoenzyme synthetase activity are all coincidental, with an apparent PI of 7.2. When SDS gel electrophoresis was carried out on the active fractions, the results shown in Fig. 13 were obtained. In order to visualize any possible minor protein bands, the gel was overloaded with the original sample which showed three major protein bands corresponding to M , = 98,000, 34,000, and 31,000. The M , = 26,000 protein observed previously in fraction 16 (Fig. 11) was not seen. A minor band of 60,000 daltons was just barely visible. Since the protein content of the fractions was low, aliquots of 0.3 ml were concentrated under vacuum to a small volume prior to treatment with the SDS buffer. The broad- ening observed in the protein bands may be due to the

FIG. 11. SDS-polyacrylamide slab gel electrophoresis. Lane 1, DNA-cellulose I1 chromatography, 6.6 pg of protein; lane 2, SDS- polyacrylamide gel electrophoresis low molecular weight protein markers; lane 3, fraction 16 of DEAE-cellulose I1 chromatography, pH 6.5,3.5 pg of protein.

der “Materials and Methods.” One ml of a DEAE-cellulose I1 prepa- FIG. 12. Chromatofocusing. The procedure was as outlined un-

ration containing 290 pg of protein was applied to a 5-ml column and eluted with polybuffer 96-acetic acid which established a pH gradient from 8-6. Biotin-binding activity (0); holoenzyme synthetase activity (A); repressor activity ( 0 , pH gradient (A).

15170 Purification and Properties of the Biotin Repressor

92.5 K - 66.2

45

31

21.5

14.4

13 0 S 14 15 16 17

FIG. 13. SDS gel electrophoresis of active fractions from chromatofocusing column. The original sample, 14.5 pg of protein (0); SDS-polyacrylamide gel electrophoresis low molecular weight markers (S ) ; fractions 13-17 with 4.1,5.2,6.0, and 2.7 pg of protein for fractions 14-17, respectively.

concentration of the polybuffer since this is not observed in the original sample which was treated in the identical manner but did not have polybuffer. I t is evident that only a single protein of M , = 34,000 is present in fractions 13-17, with the highest staining intensity in fractions 15 and 16, which also showed the highest repressor and holoenzyme synthetase activities.

DISCUSSION

Our data on the purification and properties of the biotin repressor protein have provided definitive evidence for a bi- functional protein which is capable of carrying out the follow- ing series of reactions

Biotin + ATP + repressor (R) -, (biotinyl-5"AMP.R) + P-Pi (1 )

(Biotinyl-5"AMP.R) + apoenzyme (2)

-B holoenzyme + AMP + R

(Biotinyl-5"AMP.R) + operator (0) (3)

-B (biotinyl-5"AMP. R. 0)

The synthesis of the co-repressor, biotinyl-5'-AMP, and its tight binding to the repressor protein have been previously demonstrated (5). The co-repressor-repressor complex which is the common intermediate in the above reactions can func- tion either in the synthesis of the holoenzyme, acetyl-coA carboxylase (Reaction 2) or in the repression of the biotin operon by inhibiting transcription (Reaction 3). Neutral hy- droxylamine can replace the apoenzyme as the biotin acceptor in Reaction 2 to form biotin hydroxamic acid, and we have also substantiated the formation of the biotinyl-5"AMP-re- pressor-operator complex by the [:"P]DNA fiter binding assay (5).

Enzymatic and regulator activities co-purify through the DEAE-cellulose I1 step to give an approximately 3500-fold purification. Chromatofocusing has provided a homogeneous preparation with a PI of 7.2 and possessing both activities. The biotin-binding activity, an integral part of Reaction 1, was also coincident with the two activities discussed above and was used successfully to monitor the purification of the repressor protein.

The molecular weight of the repressor protein as estimated

by SDS gel electrophoresis was 34,000, compared to an aver- age M, = 40,000 by gel exclusion chromatography. However, the possible oligomeric nature of the protein was suggested by the occasional appearance of a M , = 90,000 component on gel filtration and the appearance on native gel electrophoresis of multiple protein bands which were biologically active. A multimeric structure was also suggested by Barker et al. (10) based on intracistronic complementation analysis and on gel filtration studies of a crude extract of the holoenzyme synthe- tase enzyme which gave M , = 80,000-100,000. The conditions for the formation of the multimeric structures are presently unknown, and the structure of the protein involved in the regulatory and enzymatic activities also remains to be deter- mined. Multimeric structures of the lac and h repressor pro- teins are required for the regulation of their respective operons (19, 20).

In an earlier review on biotin (22), a parallel was drawn between the reactions involved in aminoacyl-tRNA synthesis and biotin holoenzyme synthesis, in the light of the regulatory role suggested for the aminoacyl-tRNA. It was suggested that the biotin-activating enzyme with the bound biotinyl-5"AMP may function as the co-repressor for the biotin operon. Our present study showing a single protein with repressor and holoenzyme synthetase activities provides new analogies with the amino acid systems. In both instances, an active form of the end product of a reaction sequence is involved in the regulation of its own synthesis and the synthesis of another cellular constituent. In the trp operon, the most intensively studied system (23), the trp-tRNA acts as a negative modu- lator at the attenuator site, terminating transcription of the trp operon under conditions of tryptophan excess or as a substrate for protein synthesis when the levels are low. In the biotin operon, the biotinyl-5"AMP complex either inhibits initiation of transcription by binding to the operator site or utilizes the active form of biotin for holoenzyme synthesis. The concentration of the complex which is common to both reactions will be dependent on the intracellular levels of both the apoenzyme and biotin. These in turn may be influenced by a number of factors such as growth conditions and biotin excretion. Pai and Lichtstein (24) in an early study on biotin biosynthesis, showed that the rate of synthesis of biotin and the level of bound biotin (holoenzyme) varied considerably depending on whether the cells were grown in basal or en- riched medium.

REFERENCES

1. Eisenberg, M. A. (1975) J. Bacteriol. 123, 248-254 2. Pai, C. H. (1972) J. Bacteriol. 112, 1280-1287 3. Kirschbaum, J. B., and Konrad, E. B. (1973) J. Bacteriol. 116,

4. Prakash. O., and Eisenberg, M. A. (1978) J. Bacteriol. 134, 1002-

5. Prakash, 0.. and Eisenberg, M. A. (1979) Proc. Natl. Acad. Sci.

6. Campbell, A., decampello-Campbell, A., and Chang, R. (1972)

7. Pai, C. H., and Yau, H. C. (1975) J. Microbiol. 21, 1116-1120 8. Barker, D. F., and Campbell, A. M. (1981) J. Mol. Biol. 146,451-

9. Barker, D. F., and Campbell, A. M. (1981) J. Mol. Biol. 146,469-

10. Barker, D. F., Kuhn, J., and Campbell, A. M. (1981) Gene 13.89-

11. Jobe, A., Riggs, A. D., and Bourgeois, S. (1972) J. Mol. Biol. 64,

12. Berg, P. (1956) J. Biol. Chem. 222,991-1013 13. Christner, J. F., Schlesinger, M. J., and Coon, M. J. (1964) J. Biol.

14. Lane, M. D., Young, D. L., and Lynen, F. (1964) J. Biol. Chem.

517-526

1012

U. S. A. 76,5592-5595

Proc. Natl. Acad. Sci. U. S. A. 19,676-680

467

492

120

181-199

Chem. 239,3997-4005

Purification and Properties of the Biotin Repressor 15171 239,2858-2864 227,41-44

15. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 21. Riggs, A. D., and Bourgeois, S. (1968) J. Mol. Biol. 34,361-364 16. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685 22. Eisenberg, M. A. (1973) in Advances in Enzymology (Meister, A,, 17. Layne, E. (1975) Methods Enzymol. 3,448-450 ed) Vol. 38, pp. 317-372, Interscience Publishing Inc., New York 18. Bradford, M. (1976) Anal. Biochem. 72,248-254 23. Yanofsky, C. (1981) Nature (Lond.) 289, 751-758 19. OFarrell, P. H. (1978) J. Biol. Chem. 250,4007-4021 24. Pai, C. H., and Lichstein, H. C. (1965) Biochim. Biophys. Acta 20. Pirrota, V., Chadwick, P., and Ptashne, M. (1970) Nature (Lond.) 100,28-35

15172 Purification and Properties of the Biotin Repressor Supplementary Mdterlal

to

Purification of the B i o r ~ n Repr-ess~r:

A Bifunctlanal Proteln

M d X A . Eisenberg, Prakash and Shu-Chi Hsulng

Materials and Methods

Uaterials

Phasphoceliulose (P-111 and DEAE-52 were products of the Whatman cocp. prbon&24 CI biotln. 59 mCi/lool WIL purchased from iMerSham and assayed

the cornponcnls in the &vitro assay wecc purchased from Signa Chcmlcal CO.; 8% pure by us: moat of the inpurlty (91) vas blotin-d-sulfoxide. A11 of

and the nucleoride triphosphates were treated wlfh avldin-sephaross t o

purchased from Pharnacia Fine Chemicals. A l l other chemicals were of the remove biotln contaninaflan. Chroaatofocuslnq wlybuffer and exchanger were

hlghest purity.

Calf-thymus DNA was purchased from Enro Siochem Research Products.

1. Blotln-b~ndlng Assay - A modification of the method of Jobe, Riggs, and 8ourgeols (11) was used. The assay was carried out i n a 1.5 nl centrifuge tube and the reaction mixture In 8.2 nl contarned: T r i ~ ACI. pH 1.4, 8.1 M i

mH; I1tlblotin, 8 . 5 x'l0-3M; and rebressor protein. With the more purified KC1, 0.2 M: ng A c e t a t e 4H 0 8 81 M. Wa EDTA, 1 .PI; 2-mercaptoethanol. 6 repressor preparations, 1.0 mg of 7-globulin was Included for coprecipitation. The Eontcol yntalned all Of the above components as n i l as unlabeled biotin. 8.2 x 18- M. to correc t for non-specific binding. The reaction .IXLYT~ was kept at room temperature for 5 minutes and than created with 8.8 ml of m i d S a t m a t e d ammonium siulfafe in t h e Trls buffer. The precipitate was l e f t on Ice for ten minutes and centrifuged for 5 minutes in the Cold. The supernatant fluid was carefully removed with a Pasteur pipetre and the precipitate was washed once by resunpendlng I n 1.8 -1 OC cold BIB saturated ammonium sulfate I n Trls buffer. The suspension I d 8 immediately centrifuged. the precipitate dlosolved In 0.2 nl of 0.1 N YaOH and transferred to a scintillation ulal. The Centrifuge tube was rinsed trice with 8.2 mi of llaon, and the washings rere comblned wlth the dlssolred proteln. Aquasol 2 (New England Nuclear1 111s added and the sample Counted In a Beckman Llee scintillation counter. The blotln blnding activity is expressed as plol of biotin bound per nl of enzyme.

8iotin binding was rapid, occurring In less than one minute, and the extent of binding was independent of twperature Over the range of 1O-31'. Under the condltionn of the assay, It was also Ilnear over a wide range of protein concentration. A biotin binding actlvlty Curve as a function of [14C] blotin concentration In shorn In Fig. 1 (inset). The apparent dissociation constant calculated from the s l o p oC the Scatchard plot wa3 1.3 x 18"U.

2. Blotln actlvafLon assay - The quantitative procedure of Berg 1121 Vas

hydroxdmlc acid, prepared by the method of Chcistner et . I . I131 Ber'Yed as a used to determine the formatlon of biotin hydTola.iC acld. Synthetic blotln

standard. B ~ o t i n activatlon was also measured by the holoenzyme synthetase rfTtlon whlch was a mdlfication of the method Of Lane et a1 114) UPing

The preparation of the apoenzyme will be described In a future publication. I lbiotin and acceptor protein, the apoenzyme of acetyl CoA Carboxylase.

The reactlon - 1 tu re I n 8.2 nl conta- 7.5. 8.125 Mi $4clb10t1n, 8.5 x 10 n ATP, s .n;,ngcl 5 o11. awenryme

y; potassium phosphate buffer, PA

and repressor. The reaction was carried out at 31 for2; m1nu;es and stopped by the addition of an equal volume of l8B triChloraCetlC acld containlng 2 mM biotln. After 18 minutes at room temperature, the precipitate was transferred to a 25 u mllllpole fllter f8.4Su). the content* of the tube were rinsed tvlce with 0 . 5 a1 of the preclpltatlng agent, and the washings were added to the fllter. The filter was washed

v i a l . and dried. Aama591 2 was added and the Incorwration of radloaFtiVltY three times with 2.8 m1 of 5B tclchloraCetic acid. placed in a scintillation

dete&ined by scintillation countlng. All values were corrected for the endogenous holoenzyme synthetase eccivicy present In the prtislly pnrlflsd apoenrpe preparation. The holoenrym. synthetase actively is expressed as p m 1 b i o t i n bound ~r ml of enzyme.

Electrophoresis

Polyacrylamide disc gel-electrophoresis was carried out according to the pcocedure of Dav1.S (15) u s i n g 5.6, and la cunning gels and a stacklng

CoomassiC Blue and the other Secrloned for a~tivity measurements. Each buffer of pH 7.2. The gels wece run In dupllcate; one was stained wlth

covered with 8.2 nl of the biotin-bindlng assay mixture vlth or wlthout sllce fl.l-l.5ml was placed Into a well of a tissue culture plate and

5m11 ATP. The plate was left In the refrigerator overnight and, after removlnq the slice. the Contents were tcmsferred ta a 1.5 m1 centrifuge

previourly described for the biotin bindlng assay. In the presence of ATP, tube. The ammonium sulfate precipitation and wash were carried out as

the biotinyl-5' adenylate formed rem.1"~ flrmly bound to the prcclpltafed repressor protein even at the high salt concentrations. Thls method Is 7-18 times m r e sensitive than the biotin binding assay. Vertical slab gel-electrophoresis was carried out rlth 1B running gels and the actlvity measurements sacrled out In the same manner en adjacent lanes.

Laenmll (16). Molecular rclghts were estimated vlrh the aid of the folloring SDS-Page standard markers: phosphorylase 8, 92,581 bovine Serum albumin, 66,288 ovalbumin, 45.888 carbonic anhydrase, 31,808; soybean trypsin lnhlbltor, 21,508: lysozyme, 11.490.

SDS gel-eIecLrophoresi5 was carried out according to the method of

Gel-filrrarlon

Sephacryl 5-288 column (1.6198cm) or a Biogel P-158 column (lx60cm) Molecular weights were estimated by gel-fiitratlan On either a

prevlausly calibrated rlth npproprtaf~ e s k e r s ; bovine serum albumin. 66,288; ovalbumin, 45.888; chymotrypsinogen. 25.188. The buffers used rtie elther 0.81M sodium phosphate pH 6.5. 58 glycerol with and without 8.511 NaC1 for the Bxogci c o l ~ m n and 0.81II sodium phosphate Pa 7.2-8.8; 51 glycerol with and rithout 8.5M YaCl for the Sephacryl column. The column fractions were aSlayed by the biotln binding o r the [~pPIDNA filter-binding procedures.

ChromatofoCUsinP

column washed with 18-15 bed volumes of Starting buffer. 8.825M TriS-acetate pH 8.3. A 1 -1 sample was Introduced after first running On 5 ml of the eluent polybuffer 96-acetate diluted 1 to 13 and adjusted to PO 6.0. The elutio; was continued at a rate of 30 nl/hr collecting 2 ml samples. The pH of each sample was determined with a Corning pH aeter with an expanded scale and samples were token from each fraction far activlty measurements and protein deter-ination.

A bed volume of 5nl was poured with the plybuffer exchanger and the

Isoelectric facu*ing

unit accordinq t o the reconvended proceducc of the odlnufacrucer. The PrepdcatlVe iroelectric focuslnq was carried D Y t In an LKB flat bed

proteins - r e separated on a pH gradient from 6 to 8 for 18 hrs at 15 mA 580 volts. A small portion of gel from each Slot was suspended in water pH determination. The remainder of the gel was Suspended I n buffer and

buffer and the combined filtrates were assayed f o r activity. filtered through a scintered glass disc. The filter was washed with 8.2

and LO'

m 1

PurifLCation

All steps were carried out at 4'. Proreins were determlned by the Biuret (17) or Coomassle Blue (18) procedures. Growth conditions for strain Hl85 (cIB51al. ~ 1 8 5 1 ~ 1 ~ ~ 1 8 ) and the preparation of the crude extracts were described prevlously (41 . The preparatlon 166 scaled up from 58 9.8 of frozen cells to 200-250 qms and phcnylmerhylsulfonyl fluorlde I I . ~ x I O - ~ U ) added to the breaklnq buffer.

Prota.lnc SUlfate preclpltatlon

buffer to a final concentration of about 28 m g / d and a 2B s d u t l o n Of protamlne sulfate added dropllse to a final concentrdtion af 8 . 4 B , whlle stlrrlng. The heavy preclpitatc was removed by canLrlfUqation at 15,808 r p (5534 sorvalll a d discarded.

The supernatant fluid from the crude extract was diluted wlth breaking

column Chromatography

DEAE-Cellulose I

column WaB washed thoroughly with buffer I . The 30-60B Dmmoniy. sulfate

column was washed rlth 2 literr of buffer. The column was then e l u t e d with fraction vas lntradvced into the column at the rate of 68 ml/hr and the

buffer I containlng 8 . 2 5 M lac1 and an optical denslty measurement at 280 nm and a biotin binding aoaay was carried out on each fraction. Nearly all of the blotin bindlnq actlvlty appeared In the 8.25 M NDCl fraction as shorn I n Fig. 2 (wash not shorn). Previous Studies on a small scale (PI indicated that m s t o f the activity appeared I n the buffer wash. As we scaled UP the s i z e of the prcparatlons and cecycled the DEAE-cellulose, rr found nore of the biotin binding activity elvtlng with 8.25 II MaCl. We have carried the wash and 8.25 #I NaCl fractions independently through the subsequent purification procedures and found then to behave I n an Identical manner. The sampler shoring bioiin bindlng activity were combined and precipitated rlth solid ammonium sulfate at 188 Saturatlon. The precipitate was centrifuged (130 r o t o r , Beckman1 at 29,000 r p dissolved i n a minimal a m ~ u n L of buffer I 1 (8.81 n sodium phosphate &I 6.5, 5B glycerol. and m 4 M dithiothreitol), a n i dialyzed agalnst 2 liters of the Same buffer with three changes.

Phohphocellulose

li settled bed volume of two liters of DEAE-CeII~lo5e i n a 9 Cm x 38 Cm

directions of the manufacturer and a Settled bed volume of about 1188 nl was prepared in a 9 cm I 38 C. column. The column was weshed thoroughly with buffer 11. The proteln preparatlon from the DEAE-cellulose C0iu.m was added a t a rate of 8 8 ml/hr. and the column was washed wlth 1 4 8 8 m1 of buffer

rich buffer I 1 containing first 8.25 II laC1, and then 0.5 M YaCl, collectlng collecting the wash as a single frac..on. The ~ 0 1 u m n was eluted Stepvise

blnding assay was earrfed out on every other sample. A I illustrated I n rig. 28 ml samples. The A 2 of each sample was determined and the biotin

very little was In the 0.25 II lac1 fraction. In other preparations. we were 3 . most df the biotin binding activity was i n the 0.5 #I NaCl fraction. and

able to show that in vitro repressor activity and biotin hydroranlc acid formation were alsoassociated with the biotin bindlng activity (data n o t shorn). The fractions shorlng the highest activity r e r e combined, precipitated wlth anmoniy. sulfate at 18B Saturation, and centrifuged. The

41Ycero1. 18-% DTT and 2 x I C 3 U EDTA), and dialyzed against 1 liter of the precipitate was dlSSDlYed In buffer I 1 1 ( 8 . 8 1 M rodlum phosphate pH 1.2, I8B

Whatman phorpho=ellulose (P-11) was prepared aocordlng to the

washed with buffer 111 until the A 280 was less than 0.03. One-half of the A 25 m l column (1.8x12 cm) of calf thymus DNA-~clluiose was thoroughly

phosphocellulose preparatlon (13 m 1 ) 168 applied to the column at a rate of

buffer I l l containinq 8.2 PI NaCl Eollecting 1 nl samples. The flnsl alution 38 nl/hr. The c01u.n was first washed wlth buffer and then eluted with

vas wlth 1.8 NaC1. This was the most consistent and effldent chromatographic method. a typlcal steprise elution pttern Is seen I n Fig.

A280 168 less than a . 8 3 , and the second half of the preparation wan treated 4 . The column was the; regenerated by washing rlth 1 . 8 #I NaC1 until the

i n an Identical manner The contents of the tubes sharlnq blotln bindlng activity were comblned'and the protein was precipltated with solid a u o n i u m sulfate at 11% saturation. The prsclpltatc was dissolved In buffer I l l and dialyzed against 500 m i o f the same buffer wlth three changes.

equilibrated wlth buffer I 1 contalnlnq 10B glycerol. The aCtlYe fractlons from the DNA-cellulose I1 chromatography were combined and dialylsd ag.lnSt 588 nl of buffer I1 for 2 hr. A 9 nl sample was Introduced Into the column whlch was then eluted with a gradient of buffer I1 containing 0-0.35 I4 IaC1. Twenty-five fractiOns of 3.9 m i were Collected and elutlon was eontlnusd rith the buffer containing 1.35 #I llaC1. A typlcal A 80 and aCtlY1ty profile is Shown In Fig. 6. The biotin binding and ths blotfn holoenzyme synthetss. activlties are colncidentsl. Not shorn In the flqurc 1s the repressor activity of the peak fractions 16 and 17. Por 108B repreasion of the blotln operon In the 1" eystsn 2.5 u l Of fractlon 16 O r 5 u1 of fraction 17 was required. The specific a:fivity of fraction 16 WaB also tllCe that of 11 in the biotin activation and biotin blnding assays.

.. .. . . . . ~ ~

Purification and Properties of the Biotin Repressor 15173

F l g . : B l o r i n b i n d i n g as d f u n c r l o n of b i o t l n concentration ( i n s e t ) . Scatchard p l o t ( m a i n ! . R e p r e s s o r p r o t e i n c o n c e n t r a t i o n was 3 . 8 mq/al.

:1 0.1

1

I O ! ,

P 1 9 y

K

Fraction NO