Purification and Characterization of a Membrane-bound Nonlysosomal Ceramidase from Rat Brain

Abstract

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenylSepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C 16 -ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K m of 1.29 mol % and a V max of 4.4 mol/ min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 mol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC 50 of 5-10 M. These results indicate that the purified enzyme is a novel ceramidase.Sphingolipid metabolites are now recognized as important components in signal transduction, not only in mammalian cells, but also in yeast, where they are implicated in heat stress responses. Ceramide (Cer) 1 is one of these sphingolipid metabolites, and it has been shown to play a role in apoptosis, cell cycle arrest, and differentiation (for recent reviews, see Refs. 1-3).As a consequence of this diverse biology, the study and characterization of enzymes that regulate ceramide levels has become an essential area of study. Ceramidases (CDases) are enzymes that cleave the N-acyl linkage of ceramide into sphingosine (SPH) and free fatty acid, and recent studies suggest that CDase may exert important functions in the regulation of its substrate (Cer) or in the regulation of its immediate product (SPH) or the downstream metabolite sphingosine 1-phosphate (SPP). Indeed, current understanding indicates that the major pathway for the formation of sphingosine is via the degradation of ceramide and not from the de novo pathway (4, 5). This suggests that CDases are the key enzymes to regulate levels of SPH. Two reports implicate an alkaline CDase activity in signal transduction. Using cell homogenate of rat glomerular mesangial cells, Coroneos et al. (6) have shown that an alkaline CDase activity was stimulated by the platelet-derived growth factor and not by the inflammatory cytokines (tumor necrosis factor ␣ and interleukin-1) or the vasoconstrictor peptide endothelin-1. In another report, showed in primary cultures of rat hepatocytes that alkaline CDase activity is stimulated by low concentrations of interleukin-1.

Identification of a novel amidase motif in neutral ceramidase

“…Neutral CDase was purified to homogeneity as previously described [10]. Briefly, the enzyme was extracted from the 10 000 g pellet with Triton X-100.…”

“…A membrane-bound non-lysosomal neutral CDase having a broad pH optimum has been purified to homogeneity and biochemically characterized from various mammalian tissues, including mouse liver and rat brain, kidney and intestine [9][10][11]. Using peptide sequences obtained from the purified rat brain enzyme, the human neutral CDase was cloned [12].…”

“…The enzyme exhibited Michaelis-Menten kinetics in a Triton X-100/ceramide mixed-micellar assay. Moreover, anionic phospholipids, such as PA (phosphatidic acid), PS (phosphatidylserine) and CL (cardiolipin; diphosphatidylglycerol) stimulated the activity, whereas SPH acted as a competitive inhibitor [10]. Rat brain neutral CDase has a preference for ceramide Abbreviations used: CDase, ceramidase; CL, cardiolipin (diphosphatidylglycerol); DEPC, diethyl pyrocarbonate; DFP, di-isopropyl fluorophosphate; dhCer, dihydroceramide; DTT, dithiothreitol; FAAH, fatty acid amide hydrolase; IL-1β, interleukin-1β; maCER1, mouse alkaline CDase; 2-ME, 2-mercaptoethanol; mito-CDase, mitochondrial CDase; NBD, 7-nitrobenz-2-oxa-1,3-diazole; NEM, N-ethylmaleimide; PA, phosphatidic acid; PDGF, platelet-derived growth factor; phytoCer, phytoceramide; PS, phosphatidylserine; S1P, sphingosine 1-phosphate; SPH, sphingosine; YDC1p, dihydroCDase; YPC1p, phytoCDase.…”

Bimodal Effect of Advanced Glycation End Products on Mesangial Cell Proliferation Is Mediated by Neutral Ceramidase Regulation and Endogenous Sphingolipids

“…Ceramidase Assay-Assay was adapted from El Bawab et al (36). Briefly, cells were scrapped in 300 l of cold lysis buffer (25 mM Hepes, pH 7.5, 5 mM EDTA, 0.2% Triton X-100, 1.5 mM sodium fluoride, 1 mM sodium vanadate, 10 l/ml protease inhibitors mixture (Protease Arrest™; Calbiochem)) and centrifuged for 5 min at 10,000 ϫ g at 4°C.…”

Ceramide regulates cellular homeostasis via diverse stress signaling pathways

“…A number of ceramidases have been identified in various subcellular locations including lysosomes and membranes. [60][61][62] Like ceramide, sphingosine exerts divergent effects depending on cell type. 9 Sphingosine promotes apoptosis in U937 cells 27,63,64 and prostate cancer cells, 65 induces cell arrest in Chinese hamster ovary cells, 66 and promotes proliferation of Swiss 3T3 cells.…”