ARGINASE 1 ASSAY

Arginase Assay

Background: Arginase catalyses conversion of L-arginine to urea and L-ornithine, which is a precursor of proline and polyamines. L-arginine is also a substrate of nitric oxide synthase for the formation of NO, which is produced by macrophages as a potent anti-microbial, cytotoxic and inflammatory agent. Thus arginine 1)favors cellular proliferation (polyamines) and 2) has cytostatic and cytolytic effects (NO). (High arginase would normally go together with low NO. So activated macrophages have high NO synthase activity with low arginase activity.) Protocol: 1. After removing supernatant for NO determination, wash cells with PBS and remove PBS 2. Add 50 Q l of 0.1% Triton X-100 to cells Shake at rtp for 30 min - Mix well

(can store plate now at -80rC)

3. After lysis add 50 Ql of 10 mM MnCl2, 50 mM Tris/HCl, pH 7.5. Activate for 10 min at 55rC (in incubator) 4. Transfer 25 Q l of above mix to clean eppendorf tube (store rest) Add 25 Q l of 0.5 M arginine, pH 9.7 (arginine hydrolysis) 5. Incubate at 37rC for 60 min 6. *** Stop reaction -- add 400Ql of acid mixture: H2SO4, H3PO4 and H2 O (1: 3: 7) 7. Add 25 Q l 9% ISPF (dissolve in 100% EtOH, make fresh) Heat at 100 rC for 45 min 8. 10 min in dark (or longer at room temp) 9. 200 Ql aliquotes in microtiter plate, read at 540 nm

STANDARDS: Dilute Urea 5mg/ml to 1.5 Q g/100Q l in H2O. Follow procedure as for samples from *** It is important to do the STD with the samples. Controls: No cells just reagent No Arginine + cells + reagents