title: Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase-9 and haptoglobin released by bovine granulocytes in vitro.
abstract: OBJECTIVE
  To characterize and purify covalent complexes of matrix metalloproteinase-9 (MMP-9) and haptoglobin released by bovine granulocytes in vitro.
  
  
  METHODS
  Blood samples obtained from healthy cows and cows with acute and chronic inflammation to obtain WBCs and sera.
  
  
  METHODS
  WBCs were isolated by differential centrifugation, hypotonic lysis of RBCs, and degranulated by stimulation with phorbol ester (20 ng/mL). Cell-conditioned medium was subjected to affinity and gel chromatography and purified proteins subjected to SDS- PAGE gelatin zymography, western blot analysis, Coomassie blue staining, and peptide mass spectrometry for protein identification. Sera of cows hospitalized for acute and chronic septic conditions and of clinically normal cows were analyzed with similar methods.
  
  
  RESULTS
  Matrix metalloproteinase-9 was released from neutrophils in vitro and migrated to a molecular mass of approximately 220 kd (prodimer), approximately 105 kd (promonomer), and > 220 kd (high-molecular mass complexes). These high-molecular mass complexes were composed of alpha- and beta-haptoglobin and MMP-9 (ratio13:13:1). Complexes of MMP-9 and haptoglobin had biochemical properties of both its protein constituents (i.e., enzymatic activity toward gelatin and hemoglobin binding). Complexes of MMP-9 and haptoglobin were also detected in sera of cows with acute inflammation, but not in clinically normal cows or cows with chronic disease.
  
  
  CONCLUSIONS
  A fraction of neutrophil MMP-9 is released in complex with haptoglobin. The complex is present in granules and retains biological activity of its components. Detection of the complex in serum may provide an indicator of acute inflammation.
authors.affiliation: Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Bannikov
authors.forename: John S
authors.initials: JS
authors.lastname: Mattoon
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Abrahamsen
authors.forename: Christopher
authors.initials: C
authors.lastname: Premanandan
authors.forename: Kari B
authors.initials: KB
authors.lastname: Green-Church
authors.forename: Antoinette E
authors.initials: AE
authors.lastname: Marsh
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Lakritz
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002417:Cattle / Q000097:blood* / Q000276:immunology; D002418:Cattle Diseases / Q000097:blood / Q000276:immunology*; D002846:Chromatography, Affinity / Q000662:veterinary; D002850:Chromatography, Gel / Q000662:veterinary; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D004789:Enzyme Activation; D005260:Female; D006098:Granulocytes / Q000201:enzymology* / Q000276:immunology*; D006242:Haptoglobins / Q000276:immunology* / Q000302:isolation & purification; D007249:Inflammation / Q000097:blood / Q000276:immunology / Q000662:veterinary*; D020780:Matrix Metalloproteinase 9 / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry / Q000662:veterinary
chemical_list: D006242:Haptoglobins; D020780:Matrix Metalloproteinase 9