effect of infection of the filarial parasite brugia malayi - Pondicherry ...

Reagents
1. Acetylcholine bromide : 0.1 M
2. Sodium chloride :1M
3. Magnesium chloride :IM
4. Tris-HCI buffer : 0.5 M, pH 7.5
5. EDTA : 0.2 M
6. Cocktail : Mix 13 ml of 1.0 M sodium chloride, 2 rnl of 1 M
magnesium chloride, 10 ml of 0.5 M Tris-HC1 and I0 rnl of 0.2 M EDTA.
7. Hydroxylamine hydrochloride : 2 M
8. Sodium chloride : 3.5 M
Mix reagents 7 and 8 before use in 1: 1 ratio viv (prepare freshly)
9. Hydrochloric acid :6N
10. Fenic chloride : 0.37 M in 0.1 M HCI
1 1. Reaction &urn : Mix 17.5 rnl of cocktail with 2.0 ml of 0.1 M
acetylcholine chloride and 5.5 ml of distilled water. Tlus reaction medium gives the final
concentration of 130 mM sodium chloride, 20 mM magnesium chloride, 50 mM Tris-HCI,
0.2 mM EDTA and 4 mM acetylcholine chloride.
To the reaction mehum of 0.5 ml, 0.1 rnl of tissue homogenate and 0.4 ml of
distilled water were added and the reaction mixture was incubated at 3% for 30 minutes.
Then the reaction was terminated by addmg 2.0 ml of hydroxylamine hydrochloride and
sodium chloride mixture. The contents were mixed by vortex mixer and after one minute, 1
ml of HCI was added and mixed. Then 1 ml of femc chloride was added and absorbance was
read at 540 nm. The activity of the enzyme was expressed as pmoles of acetyl choline
bromide utiliscd/min/mg protein.
4.2.1.5 7-glutamyl transpeptidase
yGTP was assayed according to the method of Orlowski and Meister (1965) and
modifid by Rosaki and Rau (1972).