ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 168, 91%921 (1973)

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Isolation of a Glucose-Binding Protein by Affinity Chromatography on

Phloretin-Agarose’

A variety of specific proteins have been iso- lated from diverse sources by affinity chromatog- raphy (1). This powerful technique should be applicable to the isolation of binding proteins (i.e., carriers) from mammalian cell membranes, and we have recently directed our efforts to the eventual isolation of the glucose carrier from the human erythrocyte. The purpose of this report is t,o demonstrate the potential usefulness of an adsorbent we have prepared as an affinity chro- matographic material for future carrier isolation work.

We anticipated that in order to selectively extract the sugar-binding protein from solubilized eryt,hrocyte membranes, an insoluble adsorbent had to be prepared which would possess a high affinity for it. n-glucose is a poor choice to serve as the ligand (affinity group) on the material. Kinetic transport studies have indicated that glucose has a low affinity for the red cell carrier recept,or site (2, 3) ; furthermore, since many pro- t,eins may have glucose-binding sites, poor spec- ificity and capacity of a support with glucose as ligand would be expected. On the other hand, the polyphenol phloretin has a much higher, specific affinity for the carrier than does glucose and it has been recognized as a potent, competitive in- hibitor of sugar transport in the erythrocyte (4,5) We have, therefore, prepared an affinity medium of insolubilized phloretin. In order to establish the capability of this new affinity material, we show here that it can separate a model glucose- binding protein from a very complex mixture in a kidney extract. We have chosen a protein which possesses an active center that binds glucose and has a very high affinity for phloretin. This phlore- tin-protein association can be competitively re- versed by free glucose (6, 7). Since mutarotation of the sugar can occur when it binds to this active site, the protein has been called a “mutarotase” (8, 9), hut its biological function remains uncer- tain. This ubiquitous protein has already been purified from beef kidney by a IO-stage procedure

1 The work was supported by Grant A.M. 06878 Met, from the U. S. Public Health Service.

using conventional techniques (10). It has a molecular weight, of about 40,000. The K, for glucose is 25 mM and the V is 2.5 X lo4 pmoles of a-n-glucose mutarotated per minute per mg pro- tein at 25’C and pH 7.4. This enzymatic activity is a useful feature and provides the final prerequisite for our affinity chromatography test system; we can readily identify this specific model protein by a polarimetric method (6) during its fractionation.

The affinity adsorbent of insolubilized phloretin was prepared by slightly modifying the procedure described by Cuatrecasas and Anfinsen (11) for coupling phenols to diazonium intermediates of agarose. Fifty-milliliter batches of lOO- to 200- mesh, 4’T0 agarose beads (Bio Rad, Richmond, CA) were activated (12) with cyanogen bromide (12.5 g) added as a solid. During the activation procedure, 8 M NaOH was used to maint,ain t,he pH at approximately 11 and ice was added to keep the mixture at approximately 2O’C. The activa- tion react,ion was allowed to proceed for about 7 min; then ice was added to the suspension and it was quickly suction-filtered on a Buchner funnel and washed with 500 ml cold 0.1 M NatCO$ (pH 10). These activated beads were mixed with 50 ml of 2 M 3,3’-diaminodipropylamine (pH lo), and the mixture was magnetically stirred overnight at, 5°C. Uncoupled amine was removed from the beads by extensive washing with wat,er and the amino alkyl derivative of the agarose (340 ml) was washed with and finally suspended in sntn- rated sodium borate. To the stirred suspension (600 ml), 5.5 g of para-nitrobenzoyl azide in 4OU ml dimethylformamide was added. After the reaction had proceeded for 8 hr at room temperature, the beads were extensively washed wit,h 50y0 dimet,hyl- formamide (v/v). Reduction of the aromatic nitro group was achieved by treatment with 0.2 .\I sodium dithionite in 0.5 M NaHCOI (pH 8.5) at 40°C for 1 hr. This reaction product was washed extensively with water, then twice with 200 ml of cold 0.5 hf HCl and finally suspended in 300 ml of 0.5 hi HCl. To the stirred suspension in an ic*cb- salt bath, 20 ml of 0.1 M NaNO% was added and diazotization was allowed t,o proceed for 7 min. The excess nitrorls acid generated in this last step

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was destroyed by the addition of urea, and then phloretin (1.1 g in 200 ml ethanol) was added to a cold, stirred suspension of 330 ml of the diazotized beads. After the addition of 100 ml of saturated NazBaO7, and as NaOH was being added to neu- tralize the mixture, the beads developed an orange-red color which intensified only slightly after several days in the cold under Nz. Approxi- mately 2 pmoles phloretin was coupled per milli- liter of beads as estimated from the decrease in uv absorbance of the mother liquor.

A crude mutarotase extract was chromato- graphed on a 120-ml column of this insolubilized phloretin with the results shown in Fig. 1. Of the total protein added, approximately 40Y0 was not tightly adsorbed but was eluted from the column as a broad band with a NaCl-NaEDTA buffer. No mutarotase was found in these fractions. This initial peak had a long trailing edge and small but measurable levels of protein were still being eluted even after almost 20 column vol of the buffer wash. Nevertheless, after 2.2 liters of collected eluate, glucose was added to the elution buffer (arrow A, Fig. 1). This caused the release of a small

protein peak which contained about 90% of the added mutarotase. A dialyzed aliquot of this peak was subjected to disc gel electrophoresis and its protein pattern (Fig. 2, II) was compared to that from pooled fractions collected before (I) and after (III) this mutarotase peak. The extreme specificity and resolving capacity of the affinity column is demonstrated by the finding that glu- cose caused the elution of only one protein as shown by the appearance of a single new band on the stained gel. This resolving power was verified by rechromatography of pooled, dialyzed fractions obtained by glucose elution from two trials. A smaller column (20 ml) of the agarose-phloretin was used for the rechromatography from which electrophoretically homogeneous protein was ob- tained by glucose elution (C, Fig. 2). Mutarotase activity on the disc gel was detected by a color test for mutarotase (14) using glucose oxidase on paper (Tes-Tape). For the test, a gel containing the electrophoresed mutarotase fraction was cut longitudinally. One half of the gel was stained with amido black lOB, and the unstained piece was checked for mutarotase activity. We found

Elution volume (liters) 3

FIG. 1. Fractionation of kidney extract on a phloretin-agarose affinity column. Beef kidney cortex (217 g) was homogenized with 270 ml 5 mM NaEDTA, pH 7.4, for 1 min in a Waring Blendor. Two centrifugations at 12,000g for 30 min and then at 100,0@3g for 90 min gave a clear supernatant fraction. This crude extract was dialyzed against 15 mM NaCl in the EDTA buffer and diluted with 6 vol of 15 mM NaCl in 5 mM NaEDTA, pH 7.4, be- fore being chromatographed. Diluted extract (122 ml; 4.1 mg protein/ml) was applied to a 2.5.cm-diam column containing 120 ml of phloretin-agarose beads which had been equil- ibrated with 15 rnM NaCl in 5 mM NaEDTA, pH 7.4. An initial large protein peak was eluted, and at the arrow marked A, the eluant was switched to one containing 500 mM glu- cose in this buffer. This was followed, at B, with 150 mM NaCl in 5 mM NaEDTA, pH 7.4 (no glucose) and at C, the eluant was 1.5 M NaCl in 5 IIIM NaEDTA, pH 7.4. Glucose was removed by dialysis and fractions I, II, III were each concentrated in an Amicon Ultra- filtration cell (PM 10 membrane) to a final volume of 12 ml before disc gel electrophoretic analysis (see Fig. 2). Protein was assayed according to the procedure described by Lowry et al. (13).

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FIG. 2. Disc gel electrophoresis of fractions from affinity chromatography of kidney extracts. (;el 13: Crude extract. Gels I., II, III represent, respectively, 20, 40, and 30 fig prot,ein from frac- tions I, IT, III in Fig. 1. The presence of the dark band in gel II which is not evident in gels I or III suggests that glucose has selectively eluted one prot)ein. (;el C (separate electrophoretic analysis) represents an aliquot (about 25 pg protein) from rechromatographed mutarotase (see text). The single detectable prot,ein in Gel C gave a positive color test for mutarotase (14) using a glucose- oxidase test system. (;els were ot)herwise stained with arnido blacak 1013.

that the stained protein band coincided with the position of mutarotase activity.

As shown in Fig. 1, additional protein (about 10’); of that applied) could be elut,ed from t,he original column by increasing the concentration of NaCl in the eluant bu8er to 150 mM and then 1.5 .M. No mutarotase act,ivit,y was detected in these two protein peaks. Their electrophoretic patterns (not shown) differed from those of earlier fract,ions.

In summary, we have demonst,rated t,hat a derivxtixed agarose, possessing phloretin as ligand, can ext,ract from a complex mixture a protein (“mutarotase”) which has characteristics similar to the human erythrocyte sugar carrier (7). Furthermore, ellltion of this protein can be achieved by simply adding to t,he eluant another ligand which rearts with the active cent,er, namely, I,-glll~osr.

Membranes from many sources have been solrlbilized by det,ergents such as Triton X-100 and sodirun deox.vcholate. Jn fart, affinity chroma-

tography syst,ems have already been sllccessfully employed (15, 16) in the presence of these de- tergents to selectively isolate specifir membrane components. That soluble phloretin interacts with the glucose carrier in the erythrocj-te men- brane is well established; the possibility t,h;rt solubilized membrane carrier can interact with insolnhilized phloret,in remains to be tested.

FRANKLIN F. F\NNIN

hN \LI) I‘-. 1 )Il~:I~l~Ic~lt

UGversitj/ of Keuluck;// College of Medicine Department of PharwLacology Lexiugtott , Kentuck!/ 40506

Received July 5, 1973

REFERENCES

1. CIJ.ZTRW.M.\S, P., .\ND ANFINS:&, c‘. B. (1971) :l,,,,‘,r. Hrv. B&hem. 40, 259-278.

2. LI:FEV~:, P. G., \ND Lr<F~<vm:, M. E. (1952) J. Get,. Physiol. 36, 891-906.

3. 8TlClN, W. 1). (1967) The Movement of Mole- cules Across Cell Membranes, p. 164, Ara- demic Press, ?;ew York.

4. LIGFI’:vK~:, P. (:. (1959) Scieuce 130, 104-105. 5. ROSFJNBI~XG, T., .\ND WILBILINDT, W. (1962)

Exp. Cell Kes. 27, 100~117. 6. DIIcDRICH, 1). F., .\NU STHINGH.\M, c‘. 11.

(1970) :lrch. Uiochem. Hiophys. 138, 493.-498. 7. l)rti:o~IcH, I>. F., .+No HTHI~vGHAM, c’. 11.

(1970) :trch,. Hiochem. Riophys. 138, 499- 505.

8. KIGLI~, I)., .\ND H\I<T~~~:I.:, 1.1. F. (1952) Bioch,em. J. 60, 341-348.

9. KI:STON, A. S. (1964) J. Hiol. (‘henl. 239, 3241 3251.

10. B\ILI.:Y, .J. M., FISHM.W, P. H., .LNI) PENT- (‘HEV, P. (; (1969) .I. Hid. Chern. 244, 781- 788.

11. CU.\TILIWIS.\S, P., \NI) ANFINXEN, C. 13. (1971) if! Methods in I’:nzymology (Cola- wick, 1;. P., and Kaplan, N. O., eds.), Vol. 22, pp. 345-378, Academic Press, ?jew York.

12. Aslrv, I<., POI~\TH, J., E:RN~,\c~<, S. (1967) h:atuw (London) 214, 1302-1304.

13. ~\I’I<Y, 0. If., 1b3l~:HROUGH, &-. d., F.\rtn, A. L., ~<.\NI).\l.l,, It. J. (1951) J. Hiol. f’hem.

193, 265-275. 14. KESTON, A. S. (1958) Fed. Pm-. Fed. .l~r.

Sot. Exp. Hiol. 17, 253 Abstr. 15. AI.I,.zN, I)., Auols.lc, .I ., .\ND CR~MP!~ON, 11. J.

(1952) Solure Sew Hiol. 236, 2%25.

16. CI;.\TI~WAS.\S, P. (1972) Proc. .y(z/. .lrcrt/. SC;. 7 T-b.4 69, 1277m 1281.