Title

Purification And Characterization Of A Novel Acid Metalloprotease From Human Articular Cartilage (proteoglycans, Degradation, Inhibitors, Osteoarthritis)

Date of Award

1986

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

Abstract

There have been several hydrolytic enzymes and proteases implicated in the degradation of cartilage matrix proteoglycans during endochondral ossification, aging, joint immobilization, rheumatoid arthritis, osteoarthritis and cartilage neoplasia. A major pathway for degradation is the action of neutral metalloprotease on proteoglycans at the physiologic pH of the matrix. We describe here, a novel metalloprotease from articular cartilage acting on proteoglycans at pH 5.3. This protease is unique in that no mammalian metalloprotease acting optimally at acidic pH has over been documented before.This thesis describes a 4400-fold purification of an acid metalloprotease to apparent homogeneity from only 20 g of human articular cartilage using: DEAE-Sephacel, AcA-54 gel filtration, Zinc-chelate-Sepharose, CBZ-Phe-TETA-Sepharose, and Chromatofocusing. The purified protease had a pH optimum of 5.3 on bovine nasal cartilage ('3)H -proteoglycan and expressed about 40% of this activity at pH 7.5. The enzyme had minimal activity on rat skin gelatin and no activity on collagen or Azocoll. It cleaved the B chain of insulin at the amino side of Leu(,15) and Leu(,17). The enzyme required 10 mM CaCl(,2) for full activity. The activity was completely inhibited by 1 mM phenanthroline in the presence of calcium; this inhibition was reversed completely by 1 mM ZnCl(,2). There was no inhibition with PMSF, iodoacetamide, oxidized glutathione or phosphoramidon. The enzyme was in a latent form and was activated with 1 mM aminophenylmercuric acetate. The latent form had M(,r) = 55,000 by SDS-gel electrophoresis and a pI = 4.95, and the APMA-activated form had M(,r) = 35,000 and a pI = 4.55. The enzyme was inhibited with a tissue inhibitor of metalloproteinase purified from human cartilage. Enzyme level was elevated three-fold in osteoarthritic tissue compared to normal tissue. A similar enzyme activity was found in placenta and in C-type granules of human leukocytes. Neutral metalloprotease and collagenase were found in specific granules. In summary, the acid metalloprotease is unique in its pH optimum. The role of this novel protease in the extracellular degradation of cartilage proteoglycans may be important during tissue hypoxia, synovial cell inflammation, or cell destruction.

Keywords

Chemistry, Biochemistry

Link to Full Text

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