InvitroSphingomyelinaseAssay

Reagents:

Lysis buffer
25 mM Tris-HCl, pH 7.4
5 mM EDTA
1 mM ATP
20 µg/ml CLAP
1 mM PMSF

Buffer A
10 mM MgCl2
0.2 M Tris-HCl, pH 7.4
0.2 % Triton X-100

Buffer B
0.2 M sodium acetate, pH 5.0
0.2 % Triton X-100

Buffer C
0.2 M Tris-HCl, pH 7.4
0.2 % Triton X-100

Isolating cellular enzyme

1) Grow 5 x 108 cells under regular growth conditions.

2) Pellet cells and wash one time with ice cold PBS.

3) Resuspend pellet in 10 ml of ice cold lysis buffer.

4) Bomb cells: 20 minutes @ 350 psi, 4°C

5) Spin lysed cell mixture at 2,100 rpm, 4°C for 10 minute.

6) Discard pellet and set aside 3 ml of supernatant.

--> Supernatant from this step = "homogenate"

7) Spin remaining supernatant (" 7 ml) @ 200,000 xg,4 °C for 30 minutes.

centrifuge ____________________
rotor ____________________
rpm ____________________
time @ plateau ____________________

8) Recover both supernatant and pellet separately.

--> Supernatant = "cytosol"

9) Resuspend pellet in 3 ml lysis buffer.

--> This fraction = "membrane"

Preparing substrate

10) Resuspend dried 14C-SM in appropriate buffer to get 20 nmoles, 2 x 105 cpm, 100 µl per sample.

Buffer A: For neutral, Mg-dependent enzyme.
Buffer B: For acidic enzyme.
Buffer C: For neutral, Mg-independent enzyme.

11) Vortex vigorously and sonicate if neccessary.

--> Make sure lipid is fully solubilized!!

Assaying enzyme activity

12) Mix cellular enzyme with inducer gently.

--> The volume of this mix should be 100 µl.

13) Pre-incubate for 10 minutes at 37 °C.

14) Add 100 µl 14C-SM mix to each sample and mix gently.

15) Incubate reaction for 15 minutes.

16) Stop reactions by adding 1.5 ml of chloroform:methanol, 1:2.

17) Vortex, add 0.2 ml of water and vortex.

18) Add 0.5 ml chloroform and vortex to break phases.

19) Add 0.5 ml water and vortex.

20) Spin samples at 3,000 rpm for 5 minutes.

21) Count 950 µl of the upper phase and 0.5 ml of the lower phase.

--> The total volume of upper, aqueous phase should equal 1.9 ml and the total volume of the lower, chloroform phase should equal 1 ml.