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Human Ovarian Carcinoma ß-N-Acetylglucosaminidase Isoenzymes and Their Role in Extracellular Matrix Degradation¹

Roswell Park Memorial Institute, Grace Cancer Drug Center, Department of Experimental Therapeutics, Buffalo, New York 14263

Degradation and invasion of basement membrane by tumor cells involves the cooperative hydrolysis of proteoglycans, collagens, and glycoproteins mediated by a number of enzymes including proteases, collagenases, and glycosidases. In order to study these processes in vitro, a tissue culture system was developed in which bovine corneal endothelial cell extracellular matrix (ECM) serves as a substrate for attachment and degradation by human ovarian carcinoma cells. Using this system, a correlation was observed between solubilization of glycoconjugates present in ECM and extracellular levels of ß-N-acetylglucosaminidase (EC 3.2.1.30). To determine the role of individual isoenzymes of ß-N-acetylglucosaminidase (ß-NAG) in ECM degradation, the cellular and secreted forms of the enzyme were fractionated and characterized. Three intracellular isoenzyme forms, A, I, and B, were isolated from invasive human ovarian carcinoma cell line A-121. In cell homogenate, forms A and B corresponded to 65 and 33% of total ß-NAG activity, respectively. Form I was found to be localized in the plasma membrane fraction of these cells. Two secreted forms of ß-NAG (A_S and B_S) were detected in serum-free medium. The separated intracellular and secreted isoenzymes demonstrated similar K_m values, ranging from 1 to 5 mM, with p-nitro-ß-N-acetylglucosaminide substrate. Treatment of [³H]glucosamine-labeled ECM with the separated isoenzymes of ß-NAG resulted in time- and concentration-dependent releases of radioactivity with potency of I > B » A. These results suggest that human ovarian carcinoma cell ß-N-acetylglucosaminidase isoenzymes (forms B and A) contribute to ECM degradation as secreted enzymes and form I as a membrane enzyme.

¹ This work was partially supported by the National Cancer Institute (Grants CA-42898 and CA-13038). Preliminary results were presented at the 79th Annual Meeting of the American Association for Cancer Research, New Orleans, May 25–28, 1988.

² To whom requests for reprints should be addressed, at Roswell Park Memorial Institute, Grace Cancer Drug Center, Department of Experimental Therapeutics, 666 Elm St., Buffalo, NY 14263.

Received 11/23/88. Revised 4/24/89. Revised 7/13/89. Accepted 7/21/89.

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