Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica.
Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.
Indra
D
D
Biomaterials Division, Central Leather Research Institute, Adyar, Chennai-600020, Tamil Nadu, India.
Ramalingam
K
K
Babu
Mary
M
D000096:Acetone / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006372:Helix, Snails; D043143:Hepatopancreas / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006909:Hydroxyproline / Q000737:chemistry; D007525:Isoelectric Focusing; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013054:Spectrophotometry, Atomic; D013696:Temperature; D013997:Time Factors; D015032:Zinc / Q000494:pharmacology
D000096:Acetone; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D015032:Zinc; D006909:Hydroxyproline