Subject(s)
Genome, Archaeal , Pyrococcus furiosus/genetics , Amino Acyl-tRNA Synthetases/chemistry , Amino Acyl-tRNA Synthetases/genetics , Amino Acyl-tRNA Synthetases/metabolism , Codon , DNA Transposable Elements , Plasmids , Pyrococcus furiosus/enzymology , Pyrococcus furiosus/physiology , Recombinant Proteins/genetics , Structure-Activity RelationshipABSTRACT
The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons.
Subject(s)
Microbial Collagenase/biosynthesis , Tetradecanoylphorbol Acetate/pharmacology , Cell Line , Enzyme Induction , Ethylmaleimide/pharmacology , Humans , Kinetics , Microbial Collagenase/isolation & purification , Microbial Collagenase/metabolism , Molecular WeightABSTRACT
Polymorphonuclear leukocytes (PMNLs) store collagenase in an inactive form in secretory granules. The enzyme can be activated in vitro by limited proteolysis or by sulfhydryl-modifying agents such as N-ethylmaleimide (NEM). We have enriched NEM-activated collagenase 820-fold using granule isolation, gel filtration, and wheat germ agglutinin (WGA)-agarose chromatography. The use of WGA-agarose resulted in a 55-fold enrichment of collagenase in a single step with very little loss of activity. The chromatographic behavior of collagenase on other lectin matrices was explored and gave information about the type of complex asparagine-linked oligosaccharide found on collagenase isolated from PMNLs.
