Biochemical and enzymatic characterization of purified covalent complexes of matrix metalloproteinase-9 and haptoglobin released by bovine granulocytes in vitro.
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.
Bannikov
Gregory A
GA
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Mattoon
John S
JS
Abrahamsen
Eric J
EJ
Premanandan
Christopher
C
Green-Church
Kari B
KB
Marsh
Antoinette E
AE
Lakritz
Jeffrey
J
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
D006242:Haptoglobins; D020780:Matrix Metalloproteinase 9