2001-2002 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Chen, WF

Qian, ZM

Xie, JX

2001-2002 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishedVoR allowe

PolyU Institutional Repository

?????????????????????????????＊陈文芳1 , 钱忠明2 , 谢俊霞1(1.???????????? ,?? ?? 266021;2.???????????????? ,??)?? ??:研究黑素转铁蛋白(p97)在家兔网织红细胞膜上的表达及其在非转铁蛋白结合铁摄取中的作用。??:聚丙烯酰胺凝胶电泳(SDS=PAGE)和放射性同位素法(59Fe)。??:①网织红细胞孵育液浓缩后 , 经 SDS-PAGE 测定 ,在分子量 97kD位置上可见一条明显的蛋白带;用磷脂酚肌醇磷脂酶 C(PI-PLC)300 mu/ml预处理网织红细胞后 ,孵育液浓缩再经 SDS-PAGE 测定 , 在分子量 97kD处仍有一条明显的蛋白带 , 且其平均 OD 值高于未经处理的网织红细胞;而成熟的红细胞在此处却没有明显的蛋白带。 ②单纯用 PI-PLC 作用于网织红细胞 ,其铁摄取无明显变化(P>0.05)。 ③去除内源性转铁蛋白后 , 再用 PI-PLC 作用网织红细胞 ,则胞浆内铁及整合到血红素中的铁均较未经处理的网织红细胞明显降低(P<0.05)。??:p97可能存在于家兔网织红细胞膜上 , 且在摄取非转铁蛋白结合铁的过程中可能发挥作用。???:　黑素转铁蛋白;　聚丙烯酚胺凝胶电泳;　非转铁蛋白结合铁;　网织红细胞?????:Q26 ?????:A ????:1000-6834(2001)04-0356-04　＊????:2000-01-10;????:2000-12-18????:???(1968-),? ,?? ,?? ,???????。　　??????(melanotransferrin)?? p97 ,????????????????????[ 1 ,2] ,???????????。???? p97????????????。???????????? ,??????-?????(GPI)??????;???p97????? ,?????????? ,???GPI 。???????????????????? 、?????? 、?? 、?? 、???????????????????? p97 ,??????? p97??? ,? p97????????????? ,??????????????。????????????? p97???? p97?????????????? 。?????????????????p97????????????? ,????????? ,?? p97?????????????????。1　?????1.1　?????59Fe(FeCI3)? Amersham ???? , ????518.13mCi/mmo1;pI-PLC? Sigma????;??????? Bio-Rad????;??????????。1.2　??????????????????????? 2? ,???? ,?????2.3? 2.7 kg ,?? Morgan?????????[ 3] 。????????????????????? ,?????????? 3 ～ 5 d ??????? 15ml ,?????0.15 mol/L NaCl???3? ,????????????? ,??? 4℃????2000 g????? 30 min ,???? 1/4??? ,???????? 78%～ 84.5%,????? PBS ??? ,??????????? 11%～ 17%。?????????????? 。?????? ,??????? ,????? 1/4 ??????? PBS??? ,????????? 11%～ 17%。1.3　???????????2×108 ?????? PI 0.5 ml???????(pH7.4)??0.5 ml?PI-PLC(300mu/ml)???????(pH 7.4)? ,30℃?? 30 min ,???? 。??? 2 000 g ?? 30 min ,????? ,? 0.2μm???? ,??? Centricon-10?? 7?? SDS ,PAGE??。?2×108 ?????? 0.5 ml???????(pH7.4)??? ,?????????? 。?????? 7??????? 10%?????????????(??????),???0.1%?????? R250??????? 30 ～ 40min ,???? 40%??? 10%????????? 1 ～ 3 h ,??????。1.4　??????????????????????59FeCl3?????? 1∶100 ?????? ,????????31.25μmol/L。356 Chin J Appl Physiol 2001;17(4)DOI :10.13459/j.cnki.cjap.2001.04.0182?????? 5?。??????? 0.1 ml???????????????????? 2.5μmol/L ,? 37℃??????? 30 min ,??????????? PBS ?? 3? ,?????? 4℃???? 2 000g ?? 10 min ,???????? 1.0ml 20 mmol/L Hepes(pH7.4)?? ,????? 4℃????19 000 g?? 10 min????? ,??????????[ 4] 。???????????? γ-?????(Packard????)??? 。???????? C(PI-PLC)???? ,? 300mu/ml PI-PLC?????????? 30 min ,????????????????? 。?????????? , ????????PBS ???? 37℃?? 30 min ,???????? ,?? 3? ,????????????PBS? ,??????????? 11%～ 17%。???? 300mu/ml PI-PLC???????? 30 min ,??????????????:???????? PBS ??30 min ,?????? 。2　??2.1　SDS-PAGE???????SDS-PAGE?????(? 1)? ,?????????? ,???? 97 kD???????????;????????????????????????? ,?????? PI-PLC??????Fig.1 Coomassie brilliant blue R250 staining profiles of SDS-PAGEof protein isolated from supernatant of cultured cells1:marker;2:Supernatant of red blood cells;3:Supernatant of reticulocytes;4:Supernatant of PI-PLC+reticulocytes????????? ,??Model GS-670(??????)????????? ,???????????(OD)????0.315? 0.350 ,?????? ,??????????????? p97。2.2　PI-PLC???????????????????? ,??? PI-PLC(300 mu/ml)???????? ,?????? PBS ?? ,????? PI-PLC??????????? PBS ??? ,??????(P >0.05);????????????????? ,???????????(P>0.05)(? 1)。????? PI-PLC????????????????? 。Tab.1 Effect of pre-treating reticulocytes withPI-PLCon Fe3+uptake( x±s , n =5)GroupIron uptake(pmol/108)Cytosol HemeControl 　　23.16±3.78 　　6.46±1.27PI-PLC 23.64±2.03 6.26±3.152.3　?????????? ,PI-PLC?????????????????????? ,??????? PI-PLC ,?????????? PBS??? ,????????(P <0.05);??????????????????? ,?????????(P <0.05)(? 2)。Tab.2 Effect of PI-PLC on Fe3+uptake of reticulocytesdepleted endogenous transferrin , ( x±s , n=5)GroupIron uptake(pmol/108)Cytosol HemeControl 　　2.35±0.37 　　1.47±0.11PI-PLC 1.82±0.23＊ 1.31±0 , 06＊　　＊P<0.05 , compared with control3　??p97?????????(???????? ,??????)???? ,?????? 。?? p97????? 、?????????????? ,????????? ,?????(GPI)????????? ,???????? ,????????????? p97????????[ 5] 。 Jefferies ?????? ,p97????????????? ,????? p97?????????????????????? ,?????????????????????[ 6] 。???????????? ,???????357????????? 2001;17(4)?????????? ,?????????????? , ????????????????[ 7 ～ 10] 。?????????????????p97?????????????????????。???? ,???????? ,?????? 7?? ,??????????????? ,?????? 97kD????????? ,?? PI-PLC(?????GPI ??? p97?????????????)??????? ,???? 97KD???????????? ,???? OD?????? ,?????????????? ,?????????????????? ,???????????????????????? ,???????? ,??????????????。????????????????????? p97。?????? ,???? p97????????? 。??2?? ,??? 300 mU/ml PI-PLC??????? ,??????????? ,?????????????[ 6] 。???????? ,????????????????????? ,?? p97?????????????。p97?????????? ,????????????????。??????????????? ,????????????。???????????? ,???????????????????? ,???????????????? ,????????????? ,?????????????????????? ,???????? ,????????????????? ,??????????????????? ,????????????????? 。?? 2 ?? ,????????????????????? , ???????90%,????????????????????????????????? ,?????????? ,?????????????????。??????? ,?? 300 mU/ml PI-PLC ???????? ,???????????????23%,??????????? 11%。????????????????????? p97 ,?????????????? ,p97????????。4　????[ 1] Dippold W G , Lioyd K O , Li L T , et al.Cell surface antigensof human melanoma:Definition of six antigenic sy stems withmonoclonal antibodies[ J] .Proc Natl Acad Sci USA , 1980 , 77:6114-6118.[ 2] FoodM R , Rothenberger S , Gabathuler R , et al.Transport andexpression in human melanomas of a trandferrin-like glyco-sylphosphatidylinositol-anchored protein[ J] .J Biolog Chem ,1994 , 269(4):3034-3040.[ 3] Qian Z M ,Morgan E H.Effect of metabolic inhibitors on up-take of non-transferrin-bound iron by reticulocytes [ J] .Biochim Biophys Acta , 1991 , 1073:456-462.[ 4] Bottomley s s ,Wolfe L C.Bridges K R.Iron metabolism inK562 erythroleukemic cells[ J] .J Biolog Chem , 1985 , 260(11):6811-6815.[ 5] Brown J P , Hewick R M , Hellstrom Ⅰ .et al.Humanmelanoma-associated antigen p97 is structurally and function-ally related to transferrin[ J] .Nature , 1982 , 296:171-173.[ 6] Jefferies W A , FoodM R, Gabathuler R , et al.Reactive mi-croglia specifically associated with amyloid plaques inAlzheimer's disease brain tissue express melanotrandferrin[ J] .Brain Research , 1996 , 712:122-126.[ 7] Hodgson L L , Quail E A , Morgan E H.Iron transport mecha-nism in reticulocytes and mature erythrocytes[ J] .J CellPhysiol , 1995 , 162:181-190.[ 8] Qian Z M , Tang p L.Mechanisms of iron uptake by mam-malian cells[ J] .Biochim Biophys Acta , 1995 , 1269:205-214.[ 9] ??? , ??? , ???.???????????????[ J] .??????????? , 1997 , 24 (1):20-25.[ 10] Kennard M L Feldmax H , Yamada T , et al.Serum level ofthe iron binding protein P97 are elevated in Alzheimer'e dis-ease[ J] .Nature Medine , 1996 , 2(11):1230-1235.THE EXPRESSION OF P97 ON THE MEMBRANE OF THERETICULOCYTE AND THE ROLE IN IRONUPTAKE IN RABBITCHEN Wen-fang1 , QIAN Zhong-ming2 , XIE Jun-xia1(1.Department of physiology , The Medical College of Qingdao University , Qingdao 266021, China;358 Chin J Appl Physiol 2001;17(4)2.Department of Applied Biology and Chemical Technology , Hong Kong Polytechnic University , China)ABSTRACTAim:To investigate the expression of p97 on the membrane of the reticulocyte and the possible role in non-transferrin bound iron uptake.Methods:The SDS-PAGE and the radioisotope(59Fe)was used in this investigation.Results:①The protein of the supernatant of reticulo-cytes was separated on SDS-PAGE.There was one band near 97kDa.The same result was obtained with reticulocytes treated with PI-PLC andthe Mean OD of the band was higher than that of untreated reticulocytes.But for the mature erythrocytes , there wasn't apparent band near97kD.②No apparent effect of only PI-PLC treatment on iron uptake by reticulocytes(P>0.05).③Reticulocytes , depleted endogenous-transferrin and then treated by PI-PLC , gave a significant decrease in iron uptake in cytosol and in heme(P <0.05).Conclusion:The re-sults support the possibility that p97 might be able to be expressed on the membrane of reticulocytes and plays a role in non-transferrin boundiron uptake by this type of cells in rabbit.　　KEY WORDS:　melanotransferrin(p97);　 SDS-PAGE;　 non-transferrin bound iron;　reticulocyte??????????????黄友章 ,汪声恒 , 杨平地 ,向　丹 , 沈建良 ,岑　坚 , 唐亚辉(???????? ,??　100037)　　????????? ,??????????????????????????。 ??????????????????????? ,????“??”????-80℃, ??????。???? , ???????? , ?????? ,??????。??? 20???????????? 15????????。1　?????(1)?? ①???????　????????(??????????12 ? , ?????????8 ?), ????(40u/ml), ????? 620(385 ～ 880)ml , 4℃? , ???? 60 min ,?????????? ,??? 220(130～ 400)ml。 4℃? , ?????????????(DMSO)、????(???? 10%, V/V)? TC-199(GIBCO)?。 ?? 2 ml/?(NUNC), ??????190(120～ 390)?。????????(1010B , CRYOMED)? , ? 1℃/min ????? 4℃??-30℃(??????????? ,?????-4℃? , ??????), ?? 10℃/min ??-80℃, ????????。??????????????(?????????????)???????????。??? ,???? ,?? 40℃??? ,???? ,?? 10%???(HS)? RPMIl640(GIBCO)?????? 10 ml?? , ?? 3? , ???????????(BMMNC)。 ②????　????????????????? ,?? BMMNC。(2)??　①??????　??????? , ????????????。 ②??????(NC)。③???????(TC)。 ④??????　? BMMNC ? 1×105/ m1·well?? , ??????????(CFU-GM)??:??? 1640 、10%HS、10%????(FCS)、0.3%??(GIBCO),????????????????????????(15%, V/V)?????????GM-CSF(100 ng/ml , ????);???????(BFU-E)????????(CFU-GEMM)??:??? IMDM(GIB-CO)、20%HS 、Epo2U/ml(????)、10%PHA-LCM(V/V , ????)、0.25%?????(GIBCO)、10-5 mol/Lα-????(GIB-CO)。???(CFU)? , ???。????????(LTC-IC):IMDM ?? 10%HS 、10%FCS 、5×10-7 mol/ L ?????(Sigma ,?? Dexter???), ?? BMMNC 1×106/ml·well , ?? 24 ??(Costar)? , 33℃, 5%CO2??? , ???????? , ? 14 d ????????? ,? X???(15 Gy)。? BMMNC ? 1×106/ml·well??????? Dexter???? , ????X ?????????? , 33℃, 5%CO2 ??????? ,????????。?? 5 ? ,?????????????? ,???????30 s , ?? IMDM ????? , ???? , ?????? , ????????? , 1 ml·well , ?? 0.3%?? , 10%HS , 10%FCS , GM-CSF 100 ng ,? IMDM ????。 14 d ?? CFU。(3)?? ①??????　????? 、??? 、?????。 ②???????　RR-NC(%)=(???÷ ???)×100。 ②????????　RR-TC(%)=(???÷ ???)×RR-NC。 ④CFU???　RR-CFU(%)=(?? CFU? ÷??CFU?)×RR-NC。 ⑤??　??????±???( x±s)?? , ????? t??。(下转第 378页)359????????? 2001;17(4)

The expression of p97 on the membrane of the reticulocyte and the role in iron uptake in rabbit

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