생명과학회지 (Journal of Life Science)

  • 제17권10호
  • /
  • Pages.1321-1329
  • /
  • 2007
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

녹용으로부터 Insulin-like Growth Factor-I의 일부정제 및 정량

Partial Purification and Quantification of Insulin-like Growth Factor-I from Red Deer Antler

  • ;
  • 모은경 (충남대학교, 농업생명과학대학, 식품공학과) ;
  • ;
  • ;
  • ;
  • 성창근 (충남대학교, 농업생명과학대학, 식품공학과)
  • Gu, Lijuan (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Mo, Eun-Kyoung (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Fang, ZheMing (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Sun, BaiShen (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Zhu, XueMei (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University) ;
  • Sung, Chang-Keun (Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University)
  • 발행 : 2007.10.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

사슴뿔은 동물세계에서 가장 빨리 성장하는 조직이다. 따라서 성장중인 사슴뿔은 뼈 성장을 촉진하는 인자가 풍부하게 포함된 것으로 생각된다. 이들 성장인자들 중 IGF-1은 뼈를 자라게 하는 조골세포의 대사에 중요한 역할을 한다고 알려져 있어 이를 정제하고자 하였다. IGF-1의 정제는 상대라고 불리는 신선한 사슴뿔을 유안침전, DEAE-Sepharose CL-6B 이온교환수지, CM-Sepharose CL-6B 양이온교환수지, Sephadex G-50의 순차적인 방법으로 할 수 있었다. 각 과정마다 IGF-1의 거동을 HPLC, SDS-PAGE, Dot blot, 그리고 western blot으로 분석하였다. IGF-1의 정량은 ELISA기술로 재조합 인간 IGF-1을 이용하여 계산되었으며, 최종 분별 액은 두 개의 단백질을 보였으나, Western-blot에서 작은 분자량인 12 kDa으로 최종 판명할 수 있었다. 정제된 단백질은 HPLC에서 retention 시간 8분만에 검출되었으며, 총 농도는 2910 ng/ml 이고 중량은 0.291 g 이었다.

Deer antler tissue contains the most rapidly growing bone in the animal kingdom. Thus, it is likely that growing antler tissue is a rich source of local paracrine bone-stimulating factors. Growth factors, at least the insulin-like growth factor (IGF), control the bone-remodelling process. In this study, we tried to isolate and purify IGF-I from fresh antler tissue by the routine isolation and purification of protein. The purification involved ammonium sulfate precipitation, DEAE-Sepharose CL-60 ion-exchange chromatography, CM-Sepharose CL-6B ion-exchange chromatography, and Sephadex G-50 chromatography. Purified fractions from each step were analyzed by high-performance liquid chromatography (HPLC), SDS polyacrylamide gel electrophoresis (SDS-PACE), Dot-blot, and Western-blot methods. Furthermore, the quantification of partially purified IGF-I was calculated by enzyme-linked immunosorbent assays (ELISA) using antibody to human recombinant IGF-1. SDS-PAGE analysis of the final fraction yielded two molecular bands and the signal band was at 12 kDa on the Western-blot film. This purified IGF-I fraction showed a peak at retention time of eight min. The quantity of IGF-I in 20 g deer antler tissue as starting weight was calculated using a standard curve to be 2910 ng/ml, and total IGF-I amount is 0.291 g. The results show that IGF-I, which can be found in deer antler, can be partially purified and quantified by classic protein isolation methods.

키워드

참고문헌

  1. Armstrong, D. G., M. J. Duclos and C. Goddard. 1990. Biological activity of insulin-like growth factor-I purified from chicken serum. Domest. Anim. Endocrinol. 7, 383-393. https://doi.org/10.1016/0739-7240(90)90043-Y
  2. Bajpai, A. and P. S. Menon. 2006. Insulin like growth factors axis and growth disorders. Indian J. Pediatr. 73, 67-71. https://doi.org/10.1007/BF02758264
  3. Ballard, F. J., R. J. Johnson, P. C. Owens, G. L. Francis, F. M. Upton, J. P. McMurtry and J. C. Wallace. 1990. Chicken insulin-like growth factor-I: amino acid sequence, radioimmunoassay, and plasma levels between strains and during growth. Gen. Comp. Endocrinol. 79, 459-468. https://doi.org/10.1016/0016-6480(90)90076-X
  4. Bauchat, J. R., W. H. Busby, Jr., A. Garmong, P. Swanson, J. Moore, M. Lin and C. Duan. 2001. Biochemical and functional analysis of a conserved IGF-binding protein isolated from rainbow trout (Oncorhynchus mykiss) hepatoma cells. J. Endocrinol. 170, 619-628. https://doi.org/10.1677/joe.0.1700619
  5. Canalis, E., T. McCarthy and M. Centrella. 1988. Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae. Endocrinology 122, 22-27. https://doi.org/10.1210/endo-122-1-22
  6. Elliott, J. L., J. M. Oldham, G. R. Ambler, J. J. Bass, G. S. Spencer, S. C. Hodgkinson, B. H. Breier, P. D. Gluckman and J. M. Suttie. 1992. Presence of insulin-like growth factor- I receptors and absence of growth hormone receptors in the antler tip. Endocrinology 130, 2513-2520. https://doi.org/10.1210/en.130.5.2513
  7. Francis, S. M. and J. M. Suttie. 1998. Detection of growth factors and proto-oncogene mRNA in the growing tip of red deer (Cervus elaphus) antler using reverse-transcriptase polymerase chain reaction (RT-PCR). J. Exp. Zool. 281, 36-42. https://doi.org/10.1002/(SICI)1097-010X(19980501)281:1<36::AID-JEZ6>3.0.CO;2-D
  8. Garcia, R. L., M. Sadighi, S. M. Francis, J. M. Suttie and J. S. Fleming. 1997. Expression of neurotrophin-3 in the growing velvet antler of the red deer Cervus elaphus. J. Mol. Endocrinol. 19, 173-182. https://doi.org/10.1677/jme.0.0190173
  9. Ghosh, S. and R. J. Playford. 2003. Bioactive natural compounds for the treatment of gastrointestinal disorders. Clin. Sci (Lond). 104, 547-556. https://doi.org/10.1042/CS20030067
  10. Gu, L. J., J. W. Wu, X. Q. Su and C. K. Sung. 2006 Isolation and Purification of Novel Anti-Fungal Peptides from Hemolymph of Immunized Larvae of Housefly, Musca domestica. J. Life Sci. 16, 387-395. https://doi.org/10.5352/JLS.2006.16.3.387
  11. Huo, Y., V. R. Schirf and W. D. Winters. 1997. The differential expression of NGFS-like substance from fresh pilose antler of Cervus nippon Temminck. Biomed. Sci. Instrum. 33, 541-543.
  12. Hwa, V., Y. Oh and R. G. Rosenfeld. 1999. The insulinlike growth factor-binding protein (IGFBP) superfamily. Endocr. Rev. 20, 761-787. https://doi.org/10.1210/er.20.6.761
  13. Li, C., D. E. Clark, E. A. Lord, J. A. Stanton and J. M. Suttie. 2002. Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery. Anat. Rec. 268, 125-130. https://doi.org/10.1002/ar.10120
  14. McElhiney, J., M. Drever, L. A. Lawton and A. J. Porter. 2002. Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water. Appl. Environ. Microbiol. 68, 5288-5295. https://doi.org/10.1128/AEM.68.11.5288-5295.2002
  15. Moore, L. G., D. Jones, M. A. Lymburn, S. C. Hodgkinson, S. R. Davis, J. M. Suttie, M. Sadighi and A. Carne. 1993. Isolation and sequencing of deer and sheep insulin-like growth factors-I and -II. Gen. Comp. Endocrinol. 92, 302-310. https://doi.org/10.1006/gcen.1993.1166
  16. Pirskanen, A., T. Jaaskelainen and P. H. Maenpaa. 1993. Insulin-like growth factor-1 modulates steroid hormone effects on osteocalcin synthesis in human MG-63 osteosarcoma cells. Eur. J. Biochem. 218, 883-891. https://doi.org/10.1111/j.1432-1033.1993.tb18444.x
  17. Price, J. S., B. O. Oyajobi, A. M. Nalin, A. Frazer, R. G. Russell and L. J. Sandell. 1996. Chondrogenesis in the regenerating antler tip in red deer: expression of collagen types I, IIA, IIB, and X demonstrated by in situ nucleic acid hybridization and immunocytochemistry. Dev. Dyn. 205, 332-347. https://doi.org/10.1002/(SICI)1097-0177(199603)205:3<332::AID-AJA12>3.0.CO;2-6
  18. Rinderknecht, E. and R. E. Humbel. 1978. The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J. Biol. Chem. 253, 2769-2776.
  19. Shimizu, M., P. Swanson, A. Hara and W. W. Dickhoff. 2003. Purification of a 41-kDa insulin-like growth factor binding protein from serum of chinook salmon, Oncorhynchus tshawytscha. Gen. Comp. Endocrinol. 132, 103-111. https://doi.org/10.1016/S0016-6480(03)00052-2
  20. Suttie, J. M., P. D. Gluckman, J. H. Butler, P. F. Fennessy, I. D. Corson and F. J. Laas. 1985. Insulin-like growth factor 1 (IGF-1) antler-stimulating hormone? Endocrinology 116, 846-848. https://doi.org/10.1210/endo-116-2-846
  21. Van den Brande, J. L., C. M. Hoogerbrugge, K. Beyreuther, P. Roepstorff, J. Jansen and S. C. van Buul-Offers. 1990. Isolation and partial characterization of IGF-like peptides from Cohn fraction IV of human plasma. Acta. Endocrinol(Copenh). 122, 683-695.

피인용 문헌

  1. Aqueous Extract of Red Deer Antler Promotes Hair Growth by Regulating the Hair Cycle and Cell Proliferation in Hair Follicles vol.2014, 2014, https://doi.org/10.1155/2014/878162