G. Chakraborty et al., MYELIN CONTAINS NEUTRAL SPHINGOMYELINASE ACTIVITY THAT IS STIMULATED BY TUMOR-NECROSIS-FACTOR-ALPHA, Journal of neuroscience research, 50(3), 1997, pp. 466-476
Purified myelin from mouse brain. was found to contain two farms of ne
utral sphingomyelinase, one Mg2+ dependent and the other Mg2+ independ
ent, The former had a pH optimum of 7.5 and K-m of 0.35 mM, whereas th
e corresponding values for the latter were pH 8.0 and K-m 3.03 mM. Spe
cific activity of the Mg2+-dependent enzyme showed a rostral-caudal gr
adient, ranging from 75 nmol/mg protein/hr in myelin from cerebral hem
ispheres to 21 nmol/mg protein/hr in myelin from spinal cord. Relative
specific activity was approximately 20% that of brain stem or cerebra
l hemisphere homogenate. Treatment of myelin with taurocholate or high
salt concentration did not significantly reduce activity of the Mg2+-
dependent enzyme. The activity of that enzyme did not change with time
or in the presence or absence of protease inhibitors; by contrast, th
at of the Mg2+-independent enzyme decreased sharply in the absence of
protease inhibitors bat rose in their presence, To test for the effect
of tumor necrosis factor-alpha (TNF alpha) on myelin sphingomyelinase
, mouse brain myelin was labeled in vivo by intracerebral injection of
[H-3]acetate into 18-20-day-old mice, After 40 hr, brain stems were r
emoved, minced, and treated with TNF alpha in Krebs-Ringer solution, a
fter which myelin was immediately isolated, Separation and counting of
individual lipids revealed TNF alpha treatment to cause increased lab
eling of myelin ceramide and cholesterol ester with concomitant decrea
se in myelin sphingomyelin. Western blotting of myelin proteins using
antibodies to the two TNF alpha receptors as probes revealed the prese
nce of the p75 receptor, Implications of these findings in relation to
possible mechanisms of autoimmune demyelination are discussed. (C) 19
97 Wiley-Liss, Inc.