Table 3.

Oligopeptidase activities in the culture supernatant, cell lysate and membrane fraction of B16F10-Nex2 cells

Cell fraction	Specific activity (μM/min per mg)	Activity per cell (pM/min per 103 cells)	Total activity (μM/min)
Supernatant	1.39 ± 0.24	36 ± 16	3.36 ± 0.15
Lysate	3.22 ± 0.96	326 ± 15	31.56 ± 0.16
Lysate + Anti-TOP Ab	0.64 ± 0.19	65 ± 5	6.31 ± 0.03
Lysate + Anti-Neurolysin Ab	2.58 ± 0.77	261 ± 5	25.25 ± 0.13
Membrane*	< 0.01	3 ± 0.5	0.2 ± 0.01

Culture supernatant, cell lysate and membrane preparation of B16F10-Nex2 cells were incubated with the fluorogenic substrate Abz-GFSPFRQ-EDDnp (20 μM) in 50 mM Tris-HCl, pH 7.4 at 37°C. Enzyme activity was followed by measuring the fluorescence at λem. = 420 nm and λex. = 320 nm. Results are expressed as means ± SD. Ab= antibodies, used at 1:300.

*Although the activity of TOP in the membrane was low, the protein was clearly detected by Western blotting with anti-TOP antibody.