Production, purification, and characterization of recombinant prohormone convertase 5 from baculovirus-infected insect cells

被引:16
作者
Cain, BM
Vishnuvardhan, D
Wang, WG
Foulon, T
Cadel, S
Cohen, P
Beinfeld, MC
机构
[1] Tufts Univ, Sch Med, Dept Pharmacol & Expt Therapeut, Boston, MA 02111 USA
[2] Inst Genet, Andover, MA 01810 USA
[3] Univ Paris 06, UMR 7631 CNRS, F-75006 Paris, France
关键词
D O I
10.1006/prep.2001.1557
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
The discovery of the prohormone convertase (PC) family of enzymes has provided several good candidates (PC1, PC2, and PC5) for the enzymes responsible for the endoproteolytic cleavage of procholecystokinin (pro-CCK). Determination of the role of individual prohormone convertases in the processing of pro-CCK is complicated because several of these enzymes are found in endocrine tumor cells expressing CCK mRNA and in identified neurons in the brain. Production of active recombinant PC5 permits the determination of its ability to cleave substrates related to pro-CCK. Active PC5, secreted from baculovirus-infected Sf9 cells, was partially purified by ion-exchange chromatography. Western blot analysis confirmed the presence of the active form of the enzyme in infected cell media and its absence from uninfected cell media. The enzyme is most active at acidic pH 6.5 and is maximally activated by 5 mM calcium. PC5 was able to cleave both monobasic and dibasic substrates without a requirement for a basic residue at P-4 and it displayed a K-m in the micromolar range. The enzyme was inhibited by EDTA, 1,10-phenanthroline, and p-CMS, as well as by two specific PC inhibitors. This is the first reported preparation of active recombinant PC5. Like the other members of its family, it has the correct catalytic characteristics in vitro to play a role in the processing of neuropeptide precursor proteins into their final bioactive forms. (C) 2002 Elsevier Science (USA).
引用
收藏
页码:227 / 233
页数:7
相关论文
共 23 条
  • [1] Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35
    Ahmad, M
    Srinivasula, SM
    Wang, LJ
    Litwack, G
    FernandesAlnemri, T
    Alnemri, ES
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (03) : 1421 - 1424
  • [2] Identification of inhibitors of prohormone convertases 1 and 2 using a peptide combinatorial library
    Apletalina, E
    Appel, J
    Lamango, NS
    Houghten, RA
    Lindberg, I
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (41) : 26589 - 26595
  • [3] PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells
    Barbero, P
    Rovère, C
    De Bie, I
    Seidah, N
    Beaudet, A
    Kitabgi, P
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (39) : 25339 - 25346
  • [4] Prohormone and proneuropeptide processing - Recent progress and future challenges
    Beinfeld, MC
    [J]. ENDOCRINE, 1998, 8 (01) : 1 - 5
  • [5] CCK biosynthesis and processing: Recent progress and future challenges
    Beinfeld, MC
    [J]. LIFE SCIENCES, 1997, 61 (24) : 2359 - 2366
  • [6] Molecular characterization, enzymatic analysis, and purification of murine proprotein convertase-1/3 (PC1/PC3) secreted from recombinant baculovirus-infected insect cells
    Boudreault, A
    Gauthier, D
    Rondeau, N
    Savaria, D
    Seidah, NG
    Chrétien, M
    Lazure, C
    [J]. PROTEIN EXPRESSION AND PURIFICATION, 1998, 14 (03) : 353 - 366
  • [7] Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3
    Brakch, N
    Rholam, M
    Simonetti, M
    Cohen, P
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 2000, 267 (06): : 1626 - 1632
  • [8] Identification and characterization of Spodoptera frugiperda furin:: A thermostable subtilisin-like endoprotease
    Cieplik, M
    Klenk, HD
    Garten, W
    [J]. BIOLOGICAL CHEMISTRY, 1998, 379 (12) : 1433 - 1440
  • [9] COHEN P, 1996, METHODS NEUROSCIENCE, P155
  • [10] DENAULT JB, 1995, J CARDIOVASC PHARM, V26, pS47