Characteristics of 92 kDa type IV collagenase/gelatinase produced by granulocytic leukemia cells: structure, expression of cDNA in E. coli and enzymic properties.
Human neutrophils can be triggered to release the collagenolytic metalloenzymes, interstitial collagenase and 92 kDa type IV collagenase/gelatinase. We have isolated and sequenced a 2.3 kb cDNA from a chronic granulocytic leukemia cDNA library that encodes for human neutrophil type IV collagenase. With the exception of one amino-acid substitution at position 280 (Arg-->Gln), the deduced amino-acid sequences of neutrophil gelatinase are identical to the amino-acid sequences of the enzyme isolated from fibrosarcoma cells. Expression of the cDNA in E. coli yielded a 72 kDa protein having a gelatinolytic activity on zymogram gel. The recombinant enzyme was activated with APMA and trypsin. The activation was accompanied by a reduction in molecular weight of approximately 10 kDa; such a reduction is characteristic of matrix metalloproteinases. The recombinant gelatinase cleaved native type V and XI collagens. Native type I collagen was not a substrate for the enzyme. These data suggest that native and recombinant 92 kDa type IV collagenase produced in E. coli have similar biochemical properties. The successful expression of the collagenase in a prokaryotic system will greatly facilitate the structure-function characterization of the enzyme and allow a more precise analysis of its physiological and pathological roles.
Pourmotabbed
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Department of Medicine, University of Tennessee at Memphis 38163.
Solomon
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Hasty
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Mainardi
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D000595:Amino Acid Sequence; D017364:Collagenases / Q000737:chemistry*; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007951:Leukemia, Myeloid / Q000201:enzymology*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D011994:Recombinant Proteins; D017364:Collagenases