Biochemical SocietyTransactions ( 1 992) 20 PARTIAL CHARACTERISATION OF BRUSHBORDER MEMBRANE BOUND IRON BINDING PROTEIN OF GUINEA PIG ENTEROCYTES.
Fig I. Sepharose C1-6B elution profile of CHAPS solubilised BBM. Opticol Density d 280 nrn
C.P.M. ( A O - ~ )
-
200
C.P.M.
. 1.4 . 1.2
T.K.BALA, E.DEBNAM and S.K.S. SRAI. 150
DEPARTMENTS OF PROTEIN AND MOLECULAR BIOLOGY AND PHYSIOLOGY, ROYAL FREE HOSPITAL SCHOOL OF MEDICINE, LONDON, NW3 2PF. Despite extensive studies, the mechanism of iron absorption is not fully understood 111. Mucosal uptake plays a role in the regulation of iron absorption. This step is thought t o be mediated by a specific carrier protein(s1, the activity and quantity of which are regulated in response t o body iron requirements [2,31. Various iron binding proteins in the intestinal mucosa or brushborder membrane (BBM) have been isolated [4,51. However, so far none have been shown to be involved in the regulation of iron absorption. In this study we describe the solubilisation and purification of an iron binding protein from guinea pig duodenal brushborder membrane. BBM vesicles were prepared from adult mucosal scrapingsfrom the duodenum of adult Dunkin-Hartley guinea pigs [6]. Vesicles were incubated with “-Fe ascorbate solution (molar ratio 1:20) at 2 l o C for 3 0 mins in order t o label iron binding protein(s1. The mixture was then solubilised with 2% CHAPS in 50mM NaCI, 20mM Hepesnris (pH6.8). The membrane suspension was then centrifuged at 150,OOOg for 1 hour and the supernatant containing solubilised protein was subjected to gel filtration (Sepharose CI-6B), ion exchange (DE-52) and SDSPAGE. Fig.1 shows the elution profile of solubilised BBM applied to Sepharose CI-6B column. Two radioactive peaks were resolved. Peak B was a non protein bound iron complex. Peak A was 59-Feassociated with protein. Fig.2 shows the elution profile of peak A applied to an ion exchange column (DE-52) and eluted in step wise salt gradient (10mM to 400mM NaCI). Three major radioactive peaks (1 t o 3) were resolved. Peaks 1 to 2 on SDS PAGE gave a number of protein bands but only one band at 1 2 0 0 K d was apparent on autoradiography. Peak 3 gave one single band at r2OOKd on silver staining and was detected by autoradiography. The unusual ion exchange profile (fig.2) of this iron binding protein is being further investigated. In conclusion we have isolated and purified an iron binding protein with a molecular weight r 2 0 0 K d which binds iron tightly in that it remains bound to the protein on SDS-PAGE. This protein is different to that described previously which has a molecular
177s
W l l W DENSITY
FRACTION NUMBER
Fig 2. Sephadex DE-52 elution profile of Peak 1. (obtained on gel filtraton C1-6B of solubilised
BBM) Opticol Density 0 280nrn
C.P.M. 14.000
optic01 density
I 2.000 ‘0
10
20
50
40
50
60
70
FRACTION NUMBER Slcpwire gradienl
lOmM TO 4WmM NoCL
mass of 50Kd [4] and loses it’s iron in SDS-PAGE. Further studies are in progress to study the role of this protein in iron uptake. 1. Conrad, M. E. (1987) Physiology of the Gastrointestinal Tract (Johnson, L. R., ed12nd Ed,pp, 1437-1453, Raven Press, New York. 2. Wheby,M.S.,Jones,L.G.,andCrosby, W.H. (1964) J. Clin. Invest. 43, 1433-1442 3. Granick. S. (1946) J. Biol. Chem. 164, 737-746 4. Teichmann,R., and Stremmel, S.,(1990) J. Clin. Invest. 86, 2145-21 52. 5. O’Donnel MW, Cox TM., (1982) Bi0chem.J. 202 102-115 6. Kessler,M., Acuto.0.. Storelli, C., Murer, H. and Muller, M.J. and Semenza,G. (1978) Biochim Biophys Acta 506, 136-154.
This study was supported by the Peter Samuel Fund.
Fig I. Sepharose C1-6B elution profile of CHAPS solubilised BBM. Opticol Density d 280 nrn
C.P.M. ( A O - ~ )
-
200
C.P.M.
. 1.4 . 1.2
T.K.BALA, E.DEBNAM and S.K.S. SRAI. 150
DEPARTMENTS OF PROTEIN AND MOLECULAR BIOLOGY AND PHYSIOLOGY, ROYAL FREE HOSPITAL SCHOOL OF MEDICINE, LONDON, NW3 2PF. Despite extensive studies, the mechanism of iron absorption is not fully understood 111. Mucosal uptake plays a role in the regulation of iron absorption. This step is thought t o be mediated by a specific carrier protein(s1, the activity and quantity of which are regulated in response t o body iron requirements [2,31. Various iron binding proteins in the intestinal mucosa or brushborder membrane (BBM) have been isolated [4,51. However, so far none have been shown to be involved in the regulation of iron absorption. In this study we describe the solubilisation and purification of an iron binding protein from guinea pig duodenal brushborder membrane. BBM vesicles were prepared from adult mucosal scrapingsfrom the duodenum of adult Dunkin-Hartley guinea pigs [6]. Vesicles were incubated with “-Fe ascorbate solution (molar ratio 1:20) at 2 l o C for 3 0 mins in order t o label iron binding protein(s1. The mixture was then solubilised with 2% CHAPS in 50mM NaCI, 20mM Hepesnris (pH6.8). The membrane suspension was then centrifuged at 150,OOOg for 1 hour and the supernatant containing solubilised protein was subjected to gel filtration (Sepharose CI-6B), ion exchange (DE-52) and SDSPAGE. Fig.1 shows the elution profile of solubilised BBM applied to Sepharose CI-6B column. Two radioactive peaks were resolved. Peak B was a non protein bound iron complex. Peak A was 59-Feassociated with protein. Fig.2 shows the elution profile of peak A applied to an ion exchange column (DE-52) and eluted in step wise salt gradient (10mM to 400mM NaCI). Three major radioactive peaks (1 t o 3) were resolved. Peaks 1 to 2 on SDS PAGE gave a number of protein bands but only one band at 1 2 0 0 K d was apparent on autoradiography. Peak 3 gave one single band at r2OOKd on silver staining and was detected by autoradiography. The unusual ion exchange profile (fig.2) of this iron binding protein is being further investigated. In conclusion we have isolated and purified an iron binding protein with a molecular weight r 2 0 0 K d which binds iron tightly in that it remains bound to the protein on SDS-PAGE. This protein is different to that described previously which has a molecular
177s
W l l W DENSITY
FRACTION NUMBER
Fig 2. Sephadex DE-52 elution profile of Peak 1. (obtained on gel filtraton C1-6B of solubilised
BBM) Opticol Density 0 280nrn
C.P.M. 14.000
optic01 density
I 2.000 ‘0
10
20
50
40
50
60
70
FRACTION NUMBER Slcpwire gradienl
lOmM TO 4WmM NoCL
mass of 50Kd [4] and loses it’s iron in SDS-PAGE. Further studies are in progress to study the role of this protein in iron uptake. 1. Conrad, M. E. (1987) Physiology of the Gastrointestinal Tract (Johnson, L. R., ed12nd Ed,pp, 1437-1453, Raven Press, New York. 2. Wheby,M.S.,Jones,L.G.,andCrosby, W.H. (1964) J. Clin. Invest. 43, 1433-1442 3. Granick. S. (1946) J. Biol. Chem. 164, 737-746 4. Teichmann,R., and Stremmel, S.,(1990) J. Clin. Invest. 86, 2145-21 52. 5. O’Donnel MW, Cox TM., (1982) Bi0chem.J. 202 102-115 6. Kessler,M., Acuto.0.. Storelli, C., Murer, H. and Muller, M.J. and Semenza,G. (1978) Biochim Biophys Acta 506, 136-154.
This study was supported by the Peter Samuel Fund.
