Human osteoclastomas contain multiple forms of cathepsin B.
During bone resorption, the osteoclast secretes hydrolytic enzymes into the sealing zone which it creates between itself and the bone surface. Since this environment is acidic, proteinases active at low pH must therefore be responsible for degrading the bone matrix, which is largely composed of type I collagen. To investigate these enzymes, we have used human osteoclastomas as the starting material. Sequential chromatography on S-Sepharose, phenyl-Sepharose, heparin-Sepharose and Sephacryl S-200HR resulted in the separation of six cysteine proteinase activities. These proteinases have Mr values ranging from 20,000 to 42,000. The pH profiles of activity showed optima between 3.5-6.0 for both synthetic substrates and type I collagen. All the proteinases were able to degrade soluble and insoluble type I collagen. The kinetics of hydrolysis using Z-Phe-Arg-NHMec and Bz-Phe-Val-Arg-NHMec as substrates resulted in values within the range expected for cathepsin B. The six activities were all inhibited by the cysteine proteinase inhibitors antipain, chymostatin, leupeptin and E-64. The rate constants of inactivation using Z-Phe-Tyr-(O-t-Bu)CHN2 were also similar to the published rates for cathepsin B. Antibodies to cathepsin B reacted with all activities. These antibodies localised the enzyme activities to the osteoclast within the tumour. Northern blotting using a cDNA probe to cathepsin B revealed three species of mRNA transcripts. These results suggest that multiple forms of cathepsin B-like proteinases are involved in osteoclastic bone resorption.
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AE
Department of Histopathology, St. George's Hospital Medical School, London, UK.
Warburton
M J
MJ
Chambers
T J
TJ
Pringle
J A
JA
Hayman
A R
AR
D000595:Amino Acid Sequence; D001859:Bone Neoplasms / Q000201:enzymology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005870:Giant Cell Tumors / Q000201:enzymology*; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010010:Osteoclasts / Q000201:enzymology; D011506:Proteins / Q000032:analysis; D013379:Substrate Specificity; D014020:Tissue Extracts / Q000032:analysis
D009842:Oligopeptides; D011506:Proteins; D014020:Tissue Extracts; D003094:Collagen; D002401:Cathepsin B