Production, Purification, and Characterization of Human Matrilysin (PUMP) from Recombinant Chinese Hamster Ovary Cells

Barnett, Jim; Straub, Kenneth; Nguyen, Bao Trâm; Chow, Jennifer; Suttman, Rebecca; Thompson, Kirstin; Tsing, Stan; Benton, Paul M. C.; Schatzman, Randall C.; Chen, M.; Chan, Hsun-Chuan

doi:10.1006/prep.1994.1004

Cited by 22 publications

(16 citation statements)

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“…An N‐terminal ubiquitin‐procollagenase‐3 fusion protein was produced in E. coli by expression from the pHC3x.2 plasmid in BL21 (DE3) cells. The expression and purification conditions were identical to those described previously for a ubiquitin‐promatrilysin protein (Barnett et al , 1994) with the following modification: the catalytic domain of collagenase‐3 (after activation by 4‐amino‐phenylmercuric acetate (APMA) such that it no longer contains the ubiquitin fusion or prodomain) was eluted from the sepharose‐Pro‐Leu‐Gly‐NHOH affinity column with 1 m NaCl, 0.1 m CAPSO, 0.1 m CaCl 2 , pH 10.0. Both prostromelysin and progelatinase B were activated by treatment with trypsin, progelatinase A was activated following incubation with APMA, conditions described by Bottomley et al (1997).…”

Section: Methodsmentioning

confidence: 99%

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Lewis

Bishop

Bottomley

et al. 1997

British J Pharmacology

129

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Section: Methodsmentioning

confidence: 99%

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Lewis

Bishop

Bottomley

et al. 1997

British J Pharmacology

129

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“…The concentration of HgC12 in the final dialysis buffer was determined to be less than 10 -12 M. Pro-HFS was activated by treatment with lmMAPMA for 16 hr, at 35~ and dialyzed as described above. Promatrilysin was kindly provided by Dr. H. E. Van Wart (Barnett et al, 1994;Sang et al, 1995). Promatrilysin was activated by treatment with 1 mM PCMB at 37~ for 2 hr and dialyzed as described above.…”

Section: Purification Of Mmpsmentioning

confidence: 99%

Activation of human progelatinase A by collagenase and matrilysin: Activation of procollagenase by matrilysin

1996

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Proteolytic and nonproteolytic methods were used to investigate the mechanism(s) by which human fibroblast progelatinase A and fibroblast-type procollagenase can be activated. Both collagenase and matrilysin were able to activate progelatinase A, resulting in an amino terminus in gelatinase A of Tyr81. The cleavage occurred distal to Cys73 within the sequence of PRCGNPDVAN80-Y81NFFPRKP. While several nonproteolytic reagents were tested, only the heavy metal Hg(II) and p-chloromercuribenzoate (PCMB) were able to induce activation of progelatinase A and resulted in the conversion of the latent 72-kDa gelatinase A to an active form of about 64.5 kDa. Matrilysin was also able to activate procollagenase and resulted in an amino terminus in collagenase of Phe81. These results suggest that fibroblast-type collagenase and matrilysin may be physiologically relevant activators of progelatinase A; the maintenance of latency and the process of activation for progelatinase A may occur through the cysteine-switch mechanism, and the proteolytic activation of procollagenase by matrilysin resulted in the same amino terminus as produced by stromelysin-1.

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“…Of the two inhibitors studied, the one with the higher solution binding constant also produced larger ion signals for the noncovalent complex in the solvent-free gas phase, which pointed to the feasibility of the use of ESI-MS for inhibitor screening studies, (J A// I Soc Mass Spectront 1995, 6, 1105-1111 M a trilysin (also referred to as pump-lor MMP-7 in the biochemical literature) belongs to the matrix metalloproteinase (MMP) family that collectively degrades connective tissue matrices [1]. MMPs are involved in normal tissue remodeling, such as growth and wound healing, and have been implicated in diseases such as arthritis and cancer [1][2][3][4][5][6][7][8][9][10][11][12]. It is known [1][2][3][4][5][6][7][8][9][10][11][12] that MMPs require zinc and calcium cations to maintain their structural integrity and biological activities, and their metal binding stoichiometries vary among the enzymes.…”

mentioning

confidence: 99%

“…MMPs are involved in normal tissue remodeling, such as growth and wound healing, and have been implicated in diseases such as arthritis and cancer [1][2][3][4][5][6][7][8][9][10][11][12]. It is known [1][2][3][4][5][6][7][8][9][10][11][12] that MMPs require zinc and calcium cations to maintain their structural integrity and biological activities, and their metal binding stoichiometries vary among the enzymes. It is essential to study the structural and functional roles of the metal cofactors and to determine their stoichiometries in the enzyme to gain an in-depth understanding of a metallo-enzyme [1][2][3][4][5][6][7][8][9][10][11][12].…”

mentioning

confidence: 99%

Study of noncovalent enzyme—inhibitor complexes and metal binding stoichiometry of matrilysin by electrospray ionization mass spectrometry

Ren

Castelhano

Billedeau

et al. 1995

J. Am. Soc. Mass Spectrom.

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Electrospray ionization mass spectrometry (ESI-MS) was used to study the noncovalent metallo-enzyme-inhibitor complexes of matrilysin (a matrix metalloproteinase of mass 18,720 u) under gentle experimental conditions and to determine the metal ion association stoichiometries in both the free enzyme and the complexes. The metal association stoichiometries of the free matrilysin were found to be highly sensitive to solution pH changes. At pH 2.2 the enzyme existed as metal-free apo-matrilysin and was not capable of binding an inhibitor. At pH 4.5-7.0 the enzyme associated specifically with zinc and calcium cations and became active in inhibitor binding. Although the stoichiometries of the metal cofactors varied (zero to two zinc and/or calcium ions) in the free enzyme dependent on solution pH, the predominant form of the enzyme-inhibitor complexes in the pH range of 4.5-7.0, in contrast, always had the metal association stoichiometry of 2Zn + 2Ca, which was the same stoichiometry the most active free metallo-enzyme had at the optimal pH of 7. At the activity onset pH of 4.5 matrilysin existed mostly as apo-enzyme (but in a conformation different from the denatured one at pH 2.2) and bound to an inhibitor slowly (time constant ∼ 2.5 min) to form the noncovalent metallo-enzyme-inhibitor complex. Of the two inhibitors studied, the one with the higher solution binding constant also produced larger ion signals for the noncovalent complex in the solvent-free gas phase, which pointed to the feasibility of the use of ESI-MS for inhibitor screening studies.

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Production, Purification, and Characterization of Human Matrilysin (PUMP) from Recombinant Chinese Hamster Ovary Cells

Cited by 22 publications

References 0 publications

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Activation of human progelatinase A by collagenase and matrilysin: Activation of procollagenase by matrilysin

Study of noncovalent enzyme—inhibitor complexes and metal binding stoichiometry of matrilysin by electrospray ionization mass spectrometry

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