Biochemical characterization of reverse activity of human recombinant neutral ceramidase

Ceramidases are enzymes that cleave the Nacyl linkage of ceramide and as a result generate sphingosine and a free fatty acid. We have purified a recombinant human neutral ceramidase from Hela cells, which is able to catalyze the reverse reaction of ceramidase i.e. generating ceramide. In order to understand the biochemistry of the human recombinant neutral ceramidase [nCDase], we have used sphingosine and C12-NBD-fatty acid as a substrate. Interestingly, this enzyme has a narrow pH optimum in the neutral range, and a very low enzyme activity is detected in the acid and alkaline range. The human nCDase is activated by Ca2+ at low concentrations, and it is inhibited by Cu2+ and Zn2+. The human recombinant nCDase is also inhibited by phospholipids such as: phosphatidic acid [PA], phosphatidyl ethanolamine [PE], phosphatidyl serine [PS], phosphatidyl choline [PC] and phosphatidyl glycerol [PG] by about 70-90% at 0.5 mM concentration. However, phosphatidyl inositol [PI] significantly stimulated the reverse activity at a very low concentration. Human nCDase was also inhibited by pyrimidine mono nucleotides such as TMP and UMP. This finding is significant as it demonstrates for the first time an effect by nucleotides on CDase activity. The enzyme is also inhibited by both oxidized and reduced GSH

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.