小牛类表皮生长因子活性肽的高分子复合物质

CALF EPIDERMAL GROWTH FACTOR ACTIVATED HIGH MOLECULAR COMPLEX

小牛颌下腺通过中性抽提，硫酸按沉淀，ＣＭ５２和ＤＥ５２负吸附，ＳｏｐｈａｄｃＸＧ７５凝胶过滤得到一个具有类表皮生长因子活性的大分子多肽，该分子可在ＰＨ７．习，ＤＥ５２层析盐梯度洗脱分开成２７ＫＤ无活性的结合蛋白和６ＫＤ的具有表皮生长因子活性小肽。通过计算，该大分子为结合蛋白和类表皮生长因子活性肽１：１结合的三聚体，大分子复合物比类表皮生长因子小肽显示更高的生物活性。

Epklermat growth boor (EGF) is a polypeptide with potent mitogeniC activity in adiversity of aam types in vivO and a widerwristy of cultured Cede in vitro.Tay1Or,et al reportedthat in crude homogenateS Of the mouse submaxillarg prepared at neutrat PH,EGF wasjSOtued as a component of a high molecule weight complex (HAW-EGF) with molecuforweight of approtoately 74KD.In this article,ac EGF actinged high molbothe complex (HMW-c-EGF) from ofsubm￣y g￣s with EGF-activity was rePOrted' 100g wet weight at submaxtheyg￣were homogeford at PH6.8 ac carried through the streptomycin snd ammonium sulfaote preCIPltuion steps' The preCipitates diSSOlVed in 300ml coal water and d￣d apbotwater the then red￣ againSt PH 5. 1,0.01M NaAc-HAc buffer the elllted with thesa!ne bUffer,￣lug the EGF-actiVe pot the adjusting the PH to 5'4 then applied toCM52 colllmn equilibrated with PHS. 4, 0. 05M NaAc-HAc buffer and eluted with the samebuffer, colleCting the Peak I the lyOPh￣. The lyop￣ sample was disSolved in PHS. 9,,0. 01M NaAc-HAc buffer the applied to sephodex G75 column and einted with the samebuffer cont￣ 0' IN Nalll, colleCting the elutes with EOF-actiWty'The mOleCular weight Of HAW-c-EGF was ￣ 100KD due to the result Of TSKG2000 SW HPLC' According to the SDS PAGE and the result Of the dissochaion Of theHMW-c-EGF, HMW-c-EGF conSisted of three c-EGF (6KD) the three c-EGFbeding Protein (27kD). The PI of c-EGF ac c-EGF bindal protein was 4. 6 and 5. 6separately on isoeleCtric focusing eleetrophoresis.The EGF-like activity Of c-EGF and HMW-c-EGF was aSSayed by the method of3H-TdR incorporation in NRK cells Both c-EGF and HMW-c-EGF Can increase tileproliferation of NRK Cell remarkable and l￣t the proliferat1On of A 431 cellS' c-EGF binding protein bed no EGF-like aCtivity.We haVe alSo COrn￣ the ativities of c-EGF. HMW-c-EGF. rh-EGF in the prolifendion Of sewrai CellS and fOUnd that HAW-C-EGF haS a ached ativity than c-EGFand rhEGF in the same conceotratlon' The rhEGF is bought from Summa' The exact rOle ofthe c-EGF bindal protein is not char.