TY - JOUR
T1 - Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells
AU - Samet, Dvorah
AU - Barenholz, Yechezkel
PY - 1999/11
Y1 - 1999/11
N2 - The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar K(m) and V(max) (~0.2 mM and ~3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37°C; (2) the aSMase is much less sensitive to Triton X-100 (>5 mM), compared with ≤0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine-rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFα, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect K(m) or V(max) of the nSMase in HL-60 sonicates. This suggests that TNFα may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed. Copyright (C) 1999 Elsevier Science Ireland Ltd.
AB - The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar K(m) and V(max) (~0.2 mM and ~3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37°C; (2) the aSMase is much less sensitive to Triton X-100 (>5 mM), compared with ≤0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine-rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFα, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect K(m) or V(max) of the nSMase in HL-60 sonicates. This suggests that TNFα may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed. Copyright (C) 1999 Elsevier Science Ireland Ltd.
KW - Acidic sphingomyelinase
KW - HL-60 cells
KW - Lissamine-rhodamine dodecanoyl sphingosyl phosphorylcholine
KW - Neutral sphingomyelinase
KW - Sphingomyelin
KW - Sphingomyelin in cycle
UR - http://www.scopus.com/inward/record.url?scp=0005226914&partnerID=8YFLogxK
U2 - 10.1016/S0009-3084(99)00076-6
DO - 10.1016/S0009-3084(99)00076-6
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C2 - 11001562
AN - SCOPUS:0005226914
SN - 0009-3084
VL - 102
SP - 65
EP - 77
JO - Chemistry and Physics of Lipids
JF - Chemistry and Physics of Lipids
IS - 1-2
ER -