| Alzheimer's disease (AD) is a kind of neuronal degeneration disease. According to the statistic, 50 percent of people who are 85 years old or high suffered from AD. In USA, the expenditure for nursing is up to 100 billion dollar in 1994. Thus, AD is now a severe social problem. Although the certain causes and the mechanism of AD are still unclear, evidence show that it is a result of various factors. It is reported that the neuron growth inhibitory factor is low in AD brain, which leads to excess consumption of neurotrophic factor, resulting neuron exhausted and die. This may be one of several intervening steps that would mediate formation of the (-amyloid and paired helical filaments.From the structure, GIF is shown to be belonged to the MT family and also designated as MT3. It is a 68-residue metalloprotein that exhibits 70% sequence identical to the MT1/2. But, different from MT1/2, MT3 is expressed exclusively in brain, and it exhibits the ability of suppressing the survival of neonatal neurons which can't be shown by MT1/2. In order to find out why they have so different functional properties, we studied the relationship between the structure, property, reaction and function of this proteins.It is difficult to isolate MT3 from the brain because of its very low concentration. So we decided to prepare it by genetic engineering method. At first, we constructed the human MT3 expression gene, and transferred it into a pGEX-4T-2 vector, then expressed it in a GST gene fusion protein system. By optimizing the conditions for thrombin cleavage and protein separation, we built up a proper separation protocol for the preparation of : GSH sephorose 4B ( Superdex 75 ( Sephadex G-25. Finally, we obtained MT3 protein with high yeild and high purity, which was identified by SDS-PAGE and ESI-MS. It has been certificated that this protocol can be used in expression and purification of various MT3 mutants without any modification, including the (-domain which was always thought to be hard to prepare.Only a few systemic research about the properties of MT3 had been recorded, and we have carried out a systematic study on it. We found that the chemical stability of MT3 is substantially lower than MT1. This can be concluded from their different association constants of both clusters. Also, we have not observed obvious two the step titration curve for MT3 which existed at the case of MT1 when titrated with acid, indicating that the stability difference of two domains is relatively small in MT3. CD spectra of acid titration showed that the metals dissociation was not a simply proceeded from one domain to other domain processly and there may be some reconstitutions of M-S bond. The result was showed even clearly by 1H-15N HSQC that in certain of pH ranges, metals dissociation can be proceeded simultaneously from two domains. The reaction activity of MT3 with small ligands also gave rise of a same conclusion. The reaction rates of MT3 with EDTA or EDNB are faster than that of MT1, indicating the clusters less stable and accessible to the solvent in MT3. In addition, when incubated with trypsin for a week, 60% of MT1 can maintain its intact structure but there is only 18% of MT3 which are intact.To clarify the cause of the difference of properties between MT3 and MT1, we studied the structure of MT3. It was shown that Cd7-MT3 is stable since there is characteristic MLCT band in both UV and CD spectra, which indicate no oxidization. And the Raman spectra also showed all the SH groups participate in chelation with metal ions, and there is no free SH or disulfide bond in the protein. But, the structure of MT3 is relatively looser than that of MT1. The results of chromatography showed that the Stoke's radius of MT3 is longer and the retain time is shorter comparing with MT1 under the same conditions. This looser structure can be explained by NMR. It has been showed that the (-domain of MT3 is relatively tight while there is very few long range NOE observed in (-domain. In (-domain, the peptide is relatively rigid except res... |
