Small Business & Entrepreneurship

A novel EP-involved pathway for iron release from soya bean seed ferritin

A novel EP-involved pathway for iron release from soya bean seed ferritin
of 13
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
  Biochem. J. (2010)  427 , 313–321 (Printed in Great Britain) doi:10.1042/BJ20100015  313 A novel EP-involved pathway for iron release from soya bean seed ferritin Xiaoping FU*, Jianjun DENG*, Haixia YANG*, Taro MASUDA † , Fumiyuki GOTO ‡ , Toshihiro YOSHIHARA ‡  and Guanghua ZHAO* 1 *CAU and ACC Joint Laboratory of Space Food, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China,  † Laboratory of Food QualityDesign and Development, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, and  ‡ BiotechnologySector, Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Chiba 270-1194, Japan Iron in phytoferritin from legume seeds is required for seedlinggerminationandearlygrowth.However,themechanismbywhichphytoferritin regulates its iron complement to these physiologicalprocesses remains unknown. In the present study, protein degrad-ation is found to occur in purified SSF (soya bean seed ferritin)(consisting of H-1 and H-2 subunits) during storage, consistentwith previous results that such degradation also occurs duringseedlinggermination.Incontrast,nodegradationisobservedwithanimal ferritin under identical conditions, suggesting that SSFautodegradation might be due to the EP (extension peptide) ontheexteriorsurfaceoftheprotein,aspecificdomainfoundonlyinphytoferritin.Indeed,EP-deletedSSFbecomesstable,confirmingthe above hypothesis. Further support comes from a proteaseactivity assay showing that EP-1 (corresponding to the EP of the H-1 subunit) exhibits significant serine protease-like activity,whereas the activity of EP-2 (corresponding to the EP of the H-2subunit) is much weaker. Consistent with the observation above,rH-1 (recombinant H-1 ferritin) is prone to degradation, whereasitsanalogue,rH-2,becomesverystableunderidenticalconditions.This demonstrates that SSF degradation mainly srcinates fromthe serine protease-like activity of EP-1. Associated with EPdegradation is a considerable increase in the rate of iron releasefrom SSF induced by ascorbate in the amyloplast (pH range, 5.8– 6.1). Thus phytoferritin may have facilitated the evolution of thespecific domain to control its iron complement in response to celliron need in the seedling stage.Key words: autodegradation, extension peptide, iron release,phytoferritin, serine protease-like activity. INTRODUCTION Ferritins are a class of multimeric iron storage and detoxificationproteins. The importance of their dual function is underscoredby their ubiquitous distribution throughout all organisms, withthe exception of fungi. The ferritin complex has 24 subunitsassembled into a spherical shell characterized by a 432-pointsymmetry. Up to 4500 Fe 3 + atoms can be stored either as thecrystalline mineral ferrihydrite or as amorphous hydrous ferricoxyphosphate in the inner cavity of the assembled ferritin shell[1–3]. Structural analyses of vertebrate, plant and bacterialferritins indicate that each subunit consists of a four-helix bundle(helices A, B, C and D) and a fifth short helix (helix E) [1,4,5].In mammals, two distinct ferritin subunits (H and L) are foundwith similar three-dimensional structures. The H subunit hasferroxidasecentresresponsibleforfastFe 2 + oxidation.Incontrast,L subunits lack ferroxidase centres, and thus do not exhibit fastFe 2 + oxidation kinetics, but facilitate nucleation of the mineralcore [5].Amino acids involved in the definition of the ferroxidase centrearestrictlyconservedinallplantferritinsexceptforPSF(peaseedferritin),whereahistidineresidueisreplacedwithaglutamicacidresidueatposition62oftheaminoacidsequenceintheferroxidasecentre [6,7]. However, plant and animal ferritins are remarkablydifferent in their cytological localization. In contrast with animalferritin existing in the cell cytoplasm, plants store iron withinferritin mainly in plastids, such as the amyloplast in seeds [3].Unlike animal ferritin, in which two types of subunits (H and L)occur,onlytheH-typesubunithasbeendescribedinphytoferritin.Ferritin from dried soya bean seed consists of two subunits, a26.5 kDa(H-1)subunitanda28.0 kDa(H-2)subunit,whichshare ∼ 80 % amino acid sequence identity [8,9]. The two subunits areencoded by two distinct genes,  SferH-1  (GenBank ® accessionnumber M64337) and  SferH-2  (GenBank ® accession number AB062754) respectively [8], and are synthesized as a precursor (32 kDa) that contains a TP (transit peptide) and a following EP(extensionpeptide)atitsN-terminal.TheTPisresponsiblefortheprecursor targeting plastids [10]. Upon transport to the plastids,the TP is cleaved from the subunit precursor, producing themature subunit which assembles in a 24-mer ferritin withinthe plastids [11]. Thus in mature phytoferritin, 24 EP domains per moleculerepresentthemajorstructuraldifferencebetweenanimaland plant ferritin. In PSF, each EP domain is composed of 24amino acid residues, 11 of which form a specific α -helix, termedthe P-helix, flanked by proline residues (X and L) [6]. Differingfrom the TP in function, it was recently discovered that the EPserves as a second binding and ferroxidase centre contributing toiron core mineralization at high ferritin iron loadings ( > 48 iron/ proteinshell)[12],indicativeoftheroleoftheEPinironoxidativedeposition in phytoferritin. Although  Arabidopsis  ferritin isconsidered crucial to protecting cells against oxidative damagerather than for iron storage [13], in legume seeds, the majorityof total iron is stored in ferritin in the amyloplast; such storageis known to meet plant demand during seedling germination andgrowth [1,14]. Therefore elucidating whether or not the EP playsaroleinphytoferritinironreleaseintheseedlingstageisthefocusof the present study.In the present study, protein degradation was found to occur uponSSF(soyabeanseedferritin)standingat4 ◦ C,anobservationconsistent with a previous report where PSF showed the same Abbreviations used: AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; AMC, 7-amino-4-methylcoumarin; Boc, t-butoxycarbonyl; EP, extension peptide;MALDI–TOF-MS, matrix-assisted laser-desorption ionization–time-of-flight MS; MCA, (7-methoxycoumarin-4-yl)acetyl; PSF, pea seed ferritin; rH-1 (rH-2),recombinant H-1 (H-2) ferritin; SSF, soya bean seed ferritin; TP, transit peptide; WT, wild-type. 1 To whom correspondence should be addressed (email c  The Authors Journal compilation  c  2010 Biochemical Society    B   i  o  c   h  e  m   i  c  a   l   J  o  u  r  n  a   l  314  X. Fu and others protein degradation taking place during seed germination [7,14].Further studies revealed that the EP of SSF has serine protease-like activity, which causes its degradation, probably because of its exposure to the exterior surface of the protein. Interestingly,removal of the EP from WT (wild-type) SSF considerablyfacilitates iron release fromferritin with thepresenceof ascorbatein the pH range 5.8–6.1, demonstrating that the EP is closelyassociated with iron release from phytoferritin. EXPERIMENTALReagents All chemicals used were of reagent grade or purer. Boc-Gln-Ala-Arg-MCA [where Boc is t-butoxycarbonyl and MCA is(7-methoxycoumarin-4-yl)acetyl] and  N  -succinyl-Ala-Phe-Lys-MCAwerepurchasedfromSigma–Aldrich.PMSF,AEBSF[4-(2-aminoethyl)benzenesulfonylfluoride],antipain,EDTA,pepstatin,benzamidine and leupeptin were from Amresco. Preparation of WT SSF, recombinant SSF and EP-deleted SSF SSF was purified [12,15] and rH-1 (recombinant H-1) prepared[8,16] as described previously. The expression plasmid for rH-2(recombinant H-2) was constructed by inserting the H-2 cDNAinto the NcoI/BamHI site of pET21d using a PCR-based method.Constructs were then introduced into the  Escherichia coli  strainBL21(DE3).Positivetransformantsofeachconstructweregrownat 37 ◦ C on an LB (Luria–Bertani) medium supplemented with50 mg/l carbenicillin, and protein expression was inducedwith 100 µ M IPTG (isopropyl  β - D -thiogalactoside) when celldensity reached an  A 600  of 0.6. Both rH-1 and rH-2 werepurified using the same methods described above for native SSF.Apoferritin was prepared as described previously [17,18]. Theconcentrations of all ferritin types were determined accordingto the Lowry method with BSA as a standard. SDS/PAGE wasperformed under reducing conditions using 15 % mini-slab gelsaccording to a previously reported method [19]. Preparation andidentification of SSF with the EP deleted was carried out asreported recently [12]. CD and fluorescence spectroscopy CD spectra were recorded on a PiStar-180 spectrometer (AppliedPhotophysics) at 25 ◦ C under a constant flow of nitrogen gas.Typically, a cell with a 0.1-cm pathlength was used for spectralmeasurements between 190 and 260 nm. The spectra representan average of 6–10 scans. CD intensities reported in the figuresare expressed in   ε  (M − 1 · cm − 1 ). Fluorescence spectra weremeasured using a Cary Eclipse spectrofluorimeter (Varian) at25 ◦ C with 280 nm as an excitation wavelength. The spectralresolution was 1.0 nm. Effect of protease inhibitors on SSF degradation Different kinds of protease inhibitors were added to theSSF solution to obtain the final concentration (5 mM PMSF,5 mM EDTA, 20 µ M pepstatin, 2 mM benzamidine and 20 µ Mleupeptin). The effective inhibiting concentrations of the proteaseinhibitors were used as described previously [20,21]. After incubation of the mixture at 4 ◦ C for 40–50 days, samples wereapplied to SDS/PAGE. A control test was performed without theaddition of inhibitors. Determination of EP protease-like activity Potential serine protease activity was determined as previouslydescribed [21,22] with slight modifications. Enzyme assaysusing peptide–MCA substrates (Boc-Gln-Ala-Arg-MCA and  N  -succinyl-Ala-Phe-Lys-MCA) were performed by fluorometricdetermination of liberated AMC (7-amino-4-methylcoumarin).Briefly, 3.94 ml of 50 mM Tris/HCl buffer solution (pH 8.0)containing 100 mM NaCl and 10 mM CaCl 2  was added to 40 µ lofpeptideMCAsubstratedissolvedinDMSO(10 mM),followedby mixing with 20 µ l of EP into a fluorescence cuvette at 25 ◦ C.TheAMCliberationbyenzymatichydrolysiswasmonitoredwitha Cary Eclipse spectrofluorimeter (Varian) at 25 ◦ C for 120 s.Fluorescence was measured using an excitation wavelength of 380 nm and an emission wavelength of 460 nm [22]. Controlsamples were created under the same conditions, except that theabove protein solution was replaced with either 20 µ l of BSA(40 µ g)or20 µ lofAlcalase(1000-folddilutionofAlcalase2.4L). Reactions between the EP and PMSF A 1 µ l aliquot of 140 mM PMSF was added to 100 µ l of EP-1(140 µ M) in 5 mM Mops at pH 7.0, and the resulting solutionwas incubated overnight at 4 ◦ C. Subsequently, the solution wasmixedwith0.1 % TFA(trifluoroaceticacid)and10 mg/mlCHCA( α -cyano-4-hydroxy cinnamic acid) prior to MALDI–TOF-MS(matrix-assisted laser-desorption ionization–time-of-flight MS)analyses.Thedegradedproductsofthe ∼ 24.5and26.5 kDaSSFsubunitswere separated electrophoretically using SDS/PAGE (15 % gels).Gel spots were prepared as described previously [9]. All massspectra of MALDI–TOF-MS were obtained on a Bruker UltraflexIII TOF/TOF (Bruker Daltonik) in positive-ion mode at anaccelerating voltage of 20 kV with a nitrogen laser (337 nm)[12]. Kinetics of iron release from holoferritin Iron release from SSF was investigated with a stopped-flowinstrument (a Hi-Tech SFA-20M apparatus) in conjunctionwith a Cary 50 spectrophotometer (Varian) using the assayprocedure described previously [23]. All concentrations statedwere final after mixing the two reagents. The mixing deadtime was determined to be 6.8 +− 0.5 ms using the DICP(dichloroindophenol) and ascorbic acid test reaction [24]. Thedevelopment of [Fe(ferrozine) 3 ] 2 + was measured by recording theincrease in absorbance at 562 nm, and the iron released estimatedusing  ε 562 = 27900 M − 1 · cm − 1 [25]. The initial rate ( ν 0 ) of ironrelease was measured as described previously [26]. Preparation and pH measurement of amyloplasts from soya beanseed cotyledons Amyloplasts were prepared according to previously reportedmethods, with slight modifications [27,28]. Soya bean seeds wereimbibed in ddH 2 O (double-distilled H 2 O) at room temperature(25 ◦ C) for 12 h in the dark and sown in a petri dish. After germination at 12, 48 and 72 h respectively, amyloplasts wereisolated from cotyledons, which were chopped with a razor bladeandimmediatelyfrozenwithliquidnitrogen.Thecotyledonsliceswere then homogenized with 10-fold ultrapure water in a WaringBlendor. This homogenate was quickly filtered through a doublelayer of nylon mesh (300 mesh). The filtrate was centrifuged at500  g  for 10 min at 4 ◦ C. The pellets were washed 6–8 times with c  The Authors Journal compilation  c  2010 Biochemical Society  EP autodegradation facilitates iron release  315 Figure 1 SDS/PAGE analysis of SSF degradation The purified SSF was incubated in buffer B at 4 ◦ C at different time intervals. Lanes 0–5correspond to incubation times of 0, 10, 20, 30, 40 and 50 days respectively. Lane M, proteinmarkers and their corresponding molecular masses. ultrapure water and were centrifuged under the same conditionsas above until the intact amyloplast was examined by lightmicroscopy (Supplementary Figure S6B). The amyloplast pelletswere crushed using an oscillator for 10 min. After centrifugationat 13200  g  for 20 min at 4 ◦ C, the pH value of the supernatant wasdetermined with a microelectrode (MI 402; Microelectrodes). RESULTSAutodegradation of SSF SSF was purified to homogeneity with an apparent molecular mass estimated to be approx. 560 kDa by native PAGE(Supplementary Figure S1A at bj4270313add.htm). Furthermore, SDS/PAGE analysis indicatedthat the ferrritin complex contained two kinds of subunits (28.0and 26.5 kDa) (Supplementary Figure S1B) present in purifiednativeferritininanapproximately1:1ratio,asreportedpreviously[8].TodeterminewhetherSSFwasstable,theproteinwasallowedtostandat4 ◦ Cfor10–50 days.SinceSSFexhibitsgoodsolubilityat pH 8.0, 50 mM phosphate buffer containing 150 mM NaCl(buffer B) at pH 8.0 was used as a sample buffer. As shown inFigure 1, two ferritin subunits began to degrade into ∼ 24.5 kDapolypeptide(s)after10 daysofincubation.Astheincubationtimeincreased from 10 to 50 days, degradation significantly increased.Similar results were obtained with other buffers, such as Mopsor Mes in the 6.0–7.5 pH range (results not shown), and proteinsamples from three different protein preparations/purifications,suggestingthattheobserveddegradationisnotsample/buffer/pH-dependent.Incontrast,suchproteindegradationwasnotobservedin animal ferritins such as HoSF (horse spleen ferritin) and HuHF(human H-chain ferritin) under the same experimental conditions(results not shown). No autodegradation with EP-deleted SSF The EP represents the major structural difference betweenplant and animal ferritins [1,8,12], raising a question as towhether the EP is involved in SSF autodegradation. To answer this question, EP-deleted SSF was prepared by incubatingWT SSF with a commercially available protease, Alcalase2.4L, at 60 ◦ C for 5 min. The newly prepared holoSSF has a Figure 2 Native PAGE and SDS/PAGE analyses of SSF with the EP deleted ( A ) Native PAGE at pH 8.3. ( B ) SDS/PAGE at pH 8.3. Lane 1, SSF with the EP deleted (molecularmass 440 kDa); lane 2, wild-type SSF (molecular mass 560 kDa); and lane M, protein markersand their corresponding molecular masses. Figure 3 Amino acid sequence of (A) WT SSF and (B) 15 N-terminalsequence residues of two subunits of SSF whose EP has been deleted byAlcalase 2.4L The EP is indicated in boldface. molecular mass of   ∼ 440 kDa (Figure 2A, lane 1) and containsthe same amount of iron within the interior core as WTholoSSF (results not shown). SDS/PAGE analysis indicated thatthe 28.0 and 26.5 kDa subunits were completely hydrolysedto two small subunits with molecular masses of 23.5 and21.0 kDa respectively (Figure 2B, lane 1). Subsequently, thefirst 15 amino acid residues of the N-terminal of the two newsubunits were determined (and are listed in Figure 3B), further confirming removal of the EP. Moreover, both WT and EP-deleted SSF had nearly the same CD (Supplementary FigureS2A at andfluorescence spectra (Supplementary Figure S2B), indicating thatthe structure of EP-deleted SSF is similar to that of WT SSF.To compare the stability of the two proteins, experiments wereconducted wherein both WT and EP-deleted SSF were allowed to c  The Authors Journal compilation  c  2010 Biochemical Society  316  X. Fu and others Figure4 ComparisonofthestabilityofWTSSFandSSFwiththeEPdeletedby SDS/PAGE analysis The samples were incubated in buffer B at 4 ◦ C for 60 days. Lane 1, holoSSF; lane 2, apoSSF;lane 3, holoSSF with the EP deleted; lane 4, apoSSF with the EP deleted; and lane M, proteinmarkers and their corresponding molecular masses. stand at 4 ◦ C over a period of 60 days. As observed above, proteindegradation occurred pronouncedly in both WT holoSSF andapoSSF (Figure 4, lanes 1 and 2), whereas EP-deleted holoSSFand apoSSF were relatively stable (Figure 4, lanes 3 and 4).This points to a functional role for the EP that is associated withproteindegradationthroughacertainway(seebelow).Evenuponstanding for 60 days or longer, no subunits with a molecular massless than 23.5 and 21.0 kDa were generated (results not shown),indicating that the EP itself is susceptible to degradation. Inaddition, WT apoSSF was also unstable, indicating that iron wasnot involved in such degradation, or at least was not a main factor. Effect of different kinds of protease inhibitors on SSF degradation The above observation raises the possibility that the EP hasprotease-like activity. To confirm this, SSF was mixed withdifferent kinds of protease inhibitors followed by standing at 4 ◦ Cfor 40 days. Final concentrations of these inhibitors were used asdescribed previously [20,21]. As shown in Figure 5(A), holoSSFdegradation was nearly completely inhibited by a serine proteaseinhibitor, PMSF, whereas EDTA (a metalloprotease inhibitor),pepstatin (an aspartic protease inhibitor) and leupeptin (a serineproteaseandcysteineproteaseinhibitor)hardlyhadanyinhibitoryeffect on the degradation. A trypsin-like serine protease inhibitor,benzamidine, did not display an inhibitory effect. Moreover, thecombination of PMSF with EDTA or other inhibitors did notexhibit an enhanced inhibitory effect. As expected, other serineprotease inhibitors, such as AEBSF and antipain, also exhibitednearly identical effects with PMSF (Supplementary Figure S3at Thus itcan be concluded from these results that the activity of serineproteases is responsible for the observed degradation of thephytoferritin EP. Nearly the same results were obtained after incubation of these protease inhibitors with SSF for 50 days at4 ◦ C (Figure 5B), further supporting this conclusion.To determine what concentration of PMSF is effective topreventSSFdegradation,aseriesofPMSFsolutionswithdifferentconcentrations were tested. As shown in Supplementary FigureS4(at,SSFdegradation was partially inactivated by 0.5 mM PMSF. Incontrast, when the PMSF concentration reached 1 mM, a nearlycomplete inhibition was obtained. Therefore 1 mM PMSF wasused during the isolation and purification of SSF. Serine protease-like activity of the EP domain of SSF To seek direct evidence of where the serine protease-like activitysrcinates from, both EP-1 with the sequence ASTVPLTGVI-FEPFEEVKKSELAVPT (corresponding to the EP from the H-1subunit) and EP-2 with the sequence ASNAPAPLAGVIFEP-FQELKKDYLAVPI (corresponding to the EP from the H-2subunit) were synthesized and their hydrolysing activity wasmeasured with Alcalase (1000-fold dilution of Alcalase 2.4L)and BSA as control samples. As expected, BSA has almostno activity against the two peptides, whereas Alcalase had thestrongest activity among all tested samples (Figure 6). Similar to BSA, EP-2 had a much weaker activity compared with theanalogue EP-1, which showed good activity against Boc-Gln-Ala-Arg-MCA (Figure 6A) and  N  -succinyl-Ala-Phe-Lys-MCA(Figure 6B), indicating that EP-1 is mainly responsible for theEP hydrolysing activity. Furthermore, it was observed thatthe combination of EP-1 and EP-2 exhibited marginally stronger activity than EP-1 alone. Similar to other serine proteases, EP-1also revealed a stronger activity against Boc-Gln-Ala-Arg-MCAthan against  N  -succinyl-Ala-Phe-Lys-MCA [21]. Although therelative catalytic activity of EP-1 is profoundly stronger than thatof EP-2, its absolute catalytic activity is still very low, excludingthe characterization of enzymology for EP-1.To gain insight into the mechanism by which PMSF preventsSSF degradation, the interaction of EP-1 with PMSF was studiedby MALDI–TOF-MS. The MS profile of the intact EP-1 showeda single peak at  m  /   z  2790.120 Da (Figure 7A). This indicatesa singly charged peptide monomer [  M  + H] + of the peptide,which is in good agreement with the predicted molecular massof 2790.240 Da. This peak rose to 2810.474 Da upon treatmentwith PMSF (Figure 7B). The difference in molecular massbetween treated EP and untreated EP is 20.354 Da, which isapproximately equal to the mass of one molecule of hydrogenfluoride (20.006 Da), which is derived from PMSF hydrolysis[29]. Therefore the observed inhibition of EP degradation byPMSF might be derived from the combination of EP-1 andhydrogen fluoride. This result provided a good explanation for why PMSF can completely inhibit SSF degradation. Comparison of stability of rH-1 and rH-2 SSF To seek further evidence of the protease activity of EP-1, thestabilities of rH-2 and rH-1 were compared by SDS/PAGEanalysis. Similar to WT SSF, rH-1 also began to degradeinto smaller polypeptides after 20 days of incubation at 25 ◦ C(Figure 8B), whereas rH-2 was very stable (Figure 8A), with nodegradation under the same conditions. This confirms the aboveconclusion that EP-1, but not EP-2, has the serine protease-likeactivity responsible for the observed SSF autodegradation duringstorage. Consistent with the present observations, MALDI–TOF-MS results showed that the peptide mass fingerprint of thedegraded product of SSF with a molecular mass of   ∼ 24.5 kDa(Figure 1) corresponded significantly with that of the 26.5 kDaSSF subunit (Supplementary Figure S5 at, as suggested by its high prob-ability-based Mowse score (131). This, once again, demonstratesthat the H-1 subunit is prone to degradation. c  The Authors Journal compilation  c  2010 Biochemical Society  EP autodegradation facilitates iron release  317 Figure 5 Effect of different kinds of protease inhibitors on SSF degradation ( A ) The purified SSF was incubated in buffer B at 4 ◦ C for 40 days; ( B ) the sample was incubated in buffer B at 4 ◦ C for 50 days. Lane 1, 5 mM EDTA; lane 2, 5 mM PMSF; lane 3, 20 µ M pepstatin;lane 4, 2 mM benzamidine; lane 5, 20 µ M leupeptin; lane 6, 5 mM EDTA and 5 mM PMSF; lane 7, 5 mM EDTA and 20 µ M pepstatin; lane 8, 5 mM EDTA, 5 mM PMSF and 20 µ M pepstatin; lane 9,control (without protease inhibitors); and lane M, protein markers and their corresponding molecular masses. Comparison of the rate of iron release from WT and EP-deletedholoSSF ToshedlightonthephysiologicalfunctionofEPautodegradation,the rate of iron release from WT holoSSF induced by ascorbicacid was compared with that from EP-deleted holoSSF. Todetermine what pH values should be used for the iron releasefrom SSF, the pH of amyloplasts isolated from SSF duringseedling germination and growth was directly measured asdescribed previously [27,28]. It was found that the amyloplastpH was in the range 5.8–6.1 (Supplementary Figure S6 at at differentgerminationperiods.ThereforethelevelofferrousatomsreleasedfromtheferritinshellwasmonitoredatpH  6.5bytheformationof the Fe 2 +  –ferrozine complex [23]. Generally, iron releasefrom EP-deleted holoSSF was much faster than that from WTholoSSF at all pH values tested (Table 1). For example, atpH 6.0, the initial rate of iron release from EP-deleted holoSSF(2.36 +− 0.15 µ M/s) is 12-fold larger than that from EP-deletedholoSSF (0.194 +− 0.013 µ M/s) (Table 1 and Figure 9). Theseresults indicate that EP removal from SSF favours iron releasefrom the protein shell to a large extent. The initial rate of ironrelease from both WT and EP-deleted SSF at pH 5.8 is smaller thanthatatpH 6.0,whichmaybeduetolargerproteinaggregationoccurring at pH 5.8 compared with at pH 6.0 (results not shown). DISCUSSION Phytoferritinisuniqueamongallknownferritinsinthatitcontainsa specific EP domain at its N-terminal sequence that could impartspecial properties to the protein [1,6,10]. Although it has beenalmost20 yearssinceEPwasfirstreported[10],itsbiologicalroleremains unknown. Until recently, the EP was reported to serve as c  The Authors Journal compilation  c  2010 Biochemical Society
Similar documents
View more...
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks