Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.
Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo rôle of gelatinases is discussed.
Hipps
D S
DS
Strangeways Research Laboratory, Cambridge, England.
Hembry
R M
RM
Docherty
A J
AJ
Reynolds
J J
JJ
Murphy
G
G
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism; D004586:Electrophoresis; D004789:Enzyme Activation; D004790:Enzyme Induction; D005347:Fibroblasts / Q000201:enzymology; D005780:Gelatin / Q000378:metabolism; D005881:Gingiva / Q000201:enzymology*; D006801:Humans; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D008666:Metalloendopeptidases / Q000378:metabolism; D003012:Microbial Collagenase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D005780:Gelatin; D003094:Collagen; D014357:Trypsin; D008666:Metalloendopeptidases; D019278:Matrix Metalloproteinase 3; D003012:Microbial Collagenase; D020780:Matrix Metalloproteinase 9