Figure 3

Enzymatic activity of nuclear-heparanase. (a) U87 glioma cells transfected with human heparanase (H-hpa), or (b) mock transfected, were lysed and fractionated to nuclear (•) and cytosolic (□) fractions and incubated (16 h, pH 5.8, 2 × 10⁶ cells) on ³⁵S-labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration on Sepharose 6B, as described in ‘Materials and Methods’. (c) Nuclear-heparanase activity in cells expressing endogenous heparanase. Nuclear (•), or cytosolic (□) fractions of human breast carcinoma cells (MDA-435) expressing endogenous human heparanase were fractionated and tested for nuclear and cytosolic heparanase enzymatic activity, as described in a. (d), Nuclear-heparanase activity following cellular uptake of recombinant heparanase. U87 glioma cells (1 × 10⁷) were incubated (DMEM, 1% FCS, 37°C, 1 h) with recombinant (65 kDa) heparanase (4 μg/ml), lysed and fractionated to nuclear (▴) and cytosolic (□) fractions, as described in ‘Materials and methods’. Following extensive washes to remove any free enzyme, fractions were incubated (pH 5.8, 37°C, 15 h) with ³⁵S-labeled ECM. The incubation medium was subjected to gel filtration on Sepharose CL-6B, as described in ‘Materials and methods’.