CONTINUOUSLY RECORDING FLUORESCENT ASSAYS OPTIMIZED FOR 5 HUMAN MATRIX METALLOPROTEINASES

被引:113
作者
NETZELARNETT, S
MALLYA, SK
NAGASE, H
BIRKEDALHANSEN, H
VANWART, HE
机构
[1] FLORIDA STATE UNIV, DEPT CHEM, TALLAHASSEE, FL 32306 USA
[2] FLORIDA STATE UNIV, INST MOLEC BIOPHYS, TALLAHASSEE, FL 32306 USA
[3] UNIV KANSAS, MED CTR, DEPT BIOCHEM & MOLEC BIOL, KANSAS CITY, KS 66103 USA
[4] UNIV ALABAMA, SCH DENT, DEPT ORAL BIOL, BIRMINGHAM, AL 35294 USA
[5] UNIV ALABAMA, SCH DENT, BIOCHEM & MOLEC BIOL RES CTR, BIRMINGHAM, AL 35294 USA
关键词
D O I
10.1016/0003-2697(91)90299-9
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Four new fluorogenic heptapeptide substrates have been synthesized with sequences that are optimized for five human matrix metalloproteinases (MMP). All four substrates are similar to one recently reported by Stack and Gray (1989, J. Biol. Chem. 264, 4277-4281) and have the fluorescent Trp residue in subsite P′2 and the dinitrophenol (DNP) quenching group on the N-terminus. The quenching of the Trp fluorescence in the intact substrate is relieved on hydrolysis of the P1-P′1 bond, giving rise to a continuously recording fluorescence assay. The residues placed in subsites P3-P′1 and P′3 have been optimized for each MMP, while Arg has been placed in P′4 to enhance solubility. Thus, DNP-Pro-Leu-Ala-Leu-Trp-Ala-Arg has been prepared as a substrate for fibroblast collagenase, DNP-Pro-Leu-Ala-Tyr-Trp-Ala-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for neutrophil collagenase, DNP-Pro-Tyr-Ala-Tyr-Trp-Met-Arg for stromelysin, and DNP-Pro-Leu-Gly-Met-Trp-Ser-Arg for both 72-kDa fibroblast gelatinase and 92-kDa neutrophil gelatinase. These substrates have been characterized with respect to their composition, solubility, optical and fluorescence spectra, and hydrolysis by their target MMP. The hydrolysis rates rival or exceed those of either their natural protein substrates or other synthetic peptides. The solubility of each substrate in assay buffer exceeds the KM value for each reaction, allowing accurate determination of the kinetic parameters. These new substrates should greatly facilitate kinetic studies of the MMP. © 1991.
引用
收藏
页码:86 / 92
页数:7
相关论文
共 50 条
  • [1] BERNHARD EJ, 1990, CANCER RES, V50, P3872
  • [2] BIRKEDALHANSEN H, 1987, METHOD ENZYMOL, V144, P140
  • [3] CHIN JR, 1985, J BIOL CHEM, V260, P2367
  • [4] COLLIER IE, 1988, J BIOL CHEM, V263, P6579
  • [5] EMONARD H, 1990, CELL MOL BIOL, V36, P131
  • [6] FIELDS GB, 1987, J BIOL CHEM, V262, P6221
  • [7] SOLID-PHASE PEPTIDE-SYNTHESIS AND SOLID-STATE NMR-SPECTROSCOPY OF [ALA3-N-15][VAL1]GRAMICIDIN-A
    FIELDS, GB
    FIELDS, CG
    PETEFISH, J
    VANWART, HE
    CROSS, TA
    [J]. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (05) : 1384 - 1388
  • [8] PROTEOLYTIC ACTIVITIES OF HUMAN FIBROBLAST COLLAGENASE - HYDROLYSIS OF A BROAD RANGE OF SUBSTRATES AT A SINGLE ACTIVE-SITE
    FIELDS, GB
    NETZELARNETT, SJ
    WINDSOR, LJ
    ENGLER, JA
    BIRKEDALHANSEN, H
    VANWART, HE
    [J]. BIOCHEMISTRY, 1990, 29 (28) : 6670 - 6677
  • [9] CLEAVAGE OF COLLAGEN TYPE-X BY HUMAN SYNOVIAL COLLAGENASE AND NEUTROPHIL ELASTASE
    GADHER, SJ
    SCHMID, TM
    HECK, LW
    WOOLLEY, DE
    [J]. MATRIX, 1989, 9 (02): : 109 - 115
  • [10] SUSCEPTIBILITY OF CARTILAGE COLLAGENS TYPE-II, TYPE-IX, TYPE-X, AND TYPE-XI TO HUMAN SYNOVIAL COLLAGENASE AND NEUTROPHIL ELASTASE
    GADHER, SJ
    EYRE, DR
    DUANCE, VC
    WOTTON, SF
    HECK, LW
    SCHMID, TM
    WOOLLEY, DE
    [J]. EUROPEAN JOURNAL OF BIOCHEMISTRY, 1988, 175 (01): : 1 - 7