Substratum acidification and proteinase activation by murine B16F10 melanoma cultures.

Abstract

Murine B16F10 melanoma cells, cultured within 0.7% agarose gels containing the fluorescent proteinase substrate acetamidofluorescein-BSA, catalyze the hydrolysis of the substrate in the region immediately surrounding the cell. Fluorescence ratio measurements on hydrolyzed substrate correlate with an average pH of 5.5 +/- 0.2 in the adjacent substratum region. Enzymatic activity within the gel is partially inhibited by leupeptin, pepstatin, phenylmethylsulfonyl fluoride. EDTA and by anti-human cathepsin B, suggesting potential roles for thiol-, aspartic- and metalloproteinases. The time-course of fluorescence intensity, correlated with substratum pH measurements, suggest that substrate hydrolysis is catalyzed by enzymes with pH optima of < 5.5. Invasion by these cells through thin barriers of reconstituted basement membrane gel (Matrigel) is totally blocked by the thiol proteinase inhibitor, leupeptin. It is suggested that secreted or cell-surface acid proteinase enzymes, activated by the cell-mediated local hyperacidity, are involved in substrate hydrolysis and that these enzymes may be important in invasiveness by this cell-line.

• Authors: P R Young, S M Spevacek
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1182 Issue 1 Pg. 69-74 (Aug 04 1993) ISSN: 0006-3002 [Print] Netherlands
• PMID: 8347688 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Fluoresceins
• Gels
• Leupeptins
• Serum Albumin, Bovine
• acetamidofluorescein
• Endopeptidases
• Cathepsin B
• leupeptin

Topics

• Animals
• Cathepsin B (analysis)
• Cell Line (enzymology)
• Endopeptidases (metabolism)
• Enzyme Activation
• Fluoresceins
• Gels
• Hydrogen-Ion Concentration
• Leupeptins (pharmacology)
• Mice
• Serum Albumin, Bovine (chemistry, metabolism)
• Tumor Cells, Cultured (enzymology)