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• W2110361141 abstract "We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibitedin vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity withd-erythro-sphingosine (Kmof 0.16 mol %, and Vmax of 0.3 μmol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Kmof 1.7 mol % and a Vmax of 0.63 μmol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified,l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5–5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid. We have previously purified a membrane-bound ceramidase from rat brain and recently cloned the human homologue. We also observed that the same enzyme is able to catalyze the reverse reaction of ceramide synthesis. To obtain insight into the biochemistry of this enzyme, we characterized in this study this reverse activity. Using sphingosine and palmitic acid as substrates, the enzyme exhibited Michaelis-Menten kinetics; however, the enzyme did not utilize palmitoyl-CoA as substrate. Also, the activity was not inhibitedin vitro and in cells by fumonisin B1, an inhibitor of the CoA-dependent ceramide synthase. The enzyme showed a narrow pH optimum in the neutral range, and there was very low activity in the alkaline range. Substrate specificity studies were performed, and the enzyme showed the highest activity withd-erythro-sphingosine (Kmof 0.16 mol %, and Vmax of 0.3 μmol/min/mg), but d-erythro-dihydrosphingosine and the three unnatural stereoisomers of sphingosine were poor substrates. The specificity for the fatty acid was also studied, and the highest activity was observed for myristic acid with a Kmof 1.7 mol % and a Vmax of 0.63 μmol/min/mg. Kinetic studies were performed to investigate the mechanism of the reaction, and Lineweaver-Burk plots indicated a sequential mechanism. Two competitive inhibitors of the two substrates were identified,l-erythro-sphingosine and myristaldehyde, and inhibition studies indicated that the reaction followed a random sequential mechanism. The effect of lipids were also tested. Most of these lipids showed moderate inhibition, whereas the effects of phosphatidic acid and cardiolipin were more potent with total inhibition at around 2.5–5 mol %. Paradoxically, cardiolipin stimulated ceramidase activity. These results define the biochemical characteristics of this reverse activity. The results are discussed in view of a possible regulation of this enzyme by the intracellular pH or by an interaction with cardiolipin and/or phosphatidic acid. ceramide ceramidase phosphatidic acid phosphatidylserine sphingomyelin sphingosine sphingosine 1-phosphate cardiolipin fumonisin B1 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. 1Hannun Y.A. Luberto C. Trends Cell. Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 2Mathias S. Pena L.A. Kolesnick R.N. Biochem. J. 1998; 335: 465-480Crossref PubMed Scopus (618) Google Scholar, 3Dickson R.C. Lester R.L. Biochim. Biophys. Acta. 1999; 1426: 347-357Crossref PubMed Scopus (168) Google Scholar). Ceramidases (CDase) are enzymes that cleave the N-acyl linkage of ceramide into sphingosine (SPH) and free fatty acid. CDases 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 (S1P). Current understanding indicates that the major pathway for the formation of sphingosine is via the degradation of ceramide and not from the de novopathway (4Merrill A.H. Wang E. Methods Enzymol. 1992; 209: 427-437Crossref PubMed Scopus (63) Google Scholar, 5Michel C. Van Echten-Deckert G. Rother J. Sandhoff K. Wang E. Merrill A.H. J. Biol. Chem. 1997; 272: 22432-22437Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). This suggests that CDases are the key enzymes to regulate levels of SPH and/or S1P. Indeed, several reports have shown the involvement of ceramidases in the regulation of Cer, SPH, and/or S1P levels in agonist-mediated cell responses. Activation of ceramidases leading to an increase of SPH and/or S1P levels and to responses associated with these lipids has been shown in rat glomerular mesangial cells stimulated with platelet-derived growth factor (6Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), in rat hepatocytes stimulated with low concentrations of interleukin 1 (7Nikolova-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merril A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), in rat mesangial cells stimulated with nitric oxide donors (8Huwiler A. Pfeilschifter J. van den Bosch H. J. Biol. Chem. 1999; 274: 7190-7195Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and in vascular smooth muscle cells treated with oxidized low density lipoprotein (9Auge N. Nikolova-Karakashian M. Carpentier S. Parthasarathy S. Negre-Salvayre A. Salvayre R. Merill Jr., A.H. Levade T. J. Biol. Chem. 1999; 274: 21533-21538Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). On the other hand, studies using inhibitors of CDases (N-oleoylethanolamine andd-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol) have also shown that inhibition of these enzymes causes an elevation in the endogenous level of ceramide, which is either sufficient to inhibit growth or augments the effects of other inducers of growth arrest (10Wiesner D.A. Kilkus J.P. Gottschalk A.R. Quintans J. Dawson G. J. Biol. Chem. 1997; 272: 9846-9868Abstract Full Text Full Text PDF Scopus (109) Google Scholar,11Bielawska A. Greenberg M.S. Perry D. Jayadev S. Shayman J.A. McKay C. Hannun Y.A. J. Biol. Chem. 1996; 271: 12646-12654Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Taken together, these observations underscore the potential importance of CDases and their roles in different process such as apoptosis and proliferation. In addition, recent studies on ceramidases have revealed the complex nature of these enzymes. In the original report on ceramidase, Gatt (12Gatt S. J. Biol. Chem. 1963; 238: 3131-3133Abstract Full Text PDF PubMed Google Scholar) proposed that a single protein catalyzes the hydrolysis of ceramide (ceramidase activity) and the reverse reaction through a CoA-independent mechanism (ceramide synthase). This intriguing observation was recently confirmed by two groups for ceramidases isolated from yeast and from mouse (13Mao C. Xu R. Bielawska A. Obeid L.M. J. Biol. Chem. 2000; 275: 6876-6884Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 14Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). We have purified and characterized a rat brain membrane-bound ceramidase (15El Bawab S. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and we recently cloned the human isoform and found that this isoform is localized to mitochondria (16El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Further studies of this enzyme also revealed that this rat brain enzyme catalyzes the reverse reaction of ceramide synthesis (16El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). To understand this enzyme, in this study, the biochemical characteristics and mechanism of action of this reverse activity were investigated. Furthermore, labeling experiments indicated that this reverse activity may account for a portion of ceramide synthesis in cells, which is not inhibitable by fumonisin B1. Biochemical characterization experiments showed specificity for the substrates and that the reaction follows a random sequential mechanism. They also suggest a possible differential regulation of the enzyme's two activities (ceramidase and reverse activity) by the intracellular pH and by the presence of cardiolipin and/or phosphatidic acid. Frozen rat brains were purchased from Pel-Freez Biologicals (Rogers, AK). Bradford protein assay was from Bio-Rad. BCA protein assay and Triton X-100 were from Pierce. [3H]Palmitoyl-CoA was from American Radiolabeled Chemicals. Lipids were from Avanti Polar Lipids. TLC plates were from Merck (Darmestad). Human embryonic kidney 293 cells overexpressing empty vector (pcDNA3.1/His) or vector containing human mitochondrial ceramidase (16El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) were cultured in minimum essential medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and 100 μg/ml Geneticin. [3H]C16-Cer, [3H]d-erythro-SPH, and [3H]d-erythro-dihydrosphingosine were synthesized as described previously (17Bielawska A. Hannun Y.A. Methods Enzymol. 2000; 311: 499-518Crossref PubMed Scopus (16) Google Scholar, 18Bielawska A. Hannun Y.A. Szulc Z. Methods Enzymol. 2000; 311: 480-498Crossref PubMed Scopus (6) Google Scholar). Ceramides with various chain length, SPH, and dihydrosphingosine were synthesized as described (19Bielawska A. Crane H.M. Liotta D. Obeid L.M. Hannun Y.A. J. Biol. Chem. 1993; 268: 26226-26232Abstract Full Text PDF PubMed Google Scholar). The purification of the protein was carried out as described previously (15El Bawab S. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, the enzyme was extracted from the 10,000 × g pellet with Triton X-100. The Triton X-100 extract was then applied to Q-Sepharose anion exchange chromatography, followed by blue-Sepharose, phenyl-Sepharose, and MonoS cation exchange chromatography. Using this protocol of purification, the specific activity was increased ∼20,000-fold, and the protein on SDS-polyacrylamide gel electrophoresis silver staining appeared as a single band in the first fractions of the last column MonoS. CDase activity was measured in a Triton X-100/Cer mixed micelle assay as described previously (15El Bawab S. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Reverse CDase activity was performed using the purified protein. Briefly, the substrates [3H]palmitic acid and SPH were first dried. The dried mixture was then resuspended by sonication in 100 μl of 200 mm Hepes buffer (pH 7) containing 0.4% of Triton X-100, and the appropriate amount of enzyme in 100 μl volume was then added. The final Triton X-100 concentration in the assay was 0.2%. The reaction was terminated by adding 2 ml of Dole solution (isopropyl alcohol/heptane/1 n NaOH, 4:1:0.1), followed by 1 ml of water and 1 ml of heptane. Under these conditions the unreacted free fatty acid remains in the aqueous/alcoholic phase. After centrifugation, the upper phase was collected, and the lower phase was washed one more time with 2 ml of heptane. The heptane phases containing the product [3H]Cer were combined and counted in liquid scintillation. Ceramide synthase (CoA-dependent) activity was assayed using rat brain microsomes as described (20Wang E. Merrill A.H. Methods Enzymol. 1999; 311: 15-21Crossref Scopus (29) Google Scholar). Briefly, the assay mixture (100 μl) contained 25 mm Tris buffer (pH 7.4), 0.5 mmdithiothreitol, 10 μm[3H]dihydrosphingosine, 200 μmpalmitoyl-CoA, and 150 μg of protein. After 30 min of incubation, the reaction was stopped by the addition of 1 ml of methanol, 0.5 ml of chloroform, and unlabeled dihydro-C16-Cer as carrier. 1 ml of chloroform and 3 ml of water were then added, and the mixture was vortexed. The aqueous layer was then discarded, and the chloroform layer was dried and applied on TLC, and lipids were separated using the solvent mixture ethyl acetate:isooctane:acetic acid (50/50/10, v/v). The dihydroceramide band was then scraped and counted. Protein concentration was determined using the Bradford assay or the BCA assay in samples containing Triton X-100. In experiments studying the SPH specificity, the assay contained [3H]palmitic acid at saturating concentration of 16 mol %; the assay in these experiments was performed as described above. In experiments studying the fatty acid chain specificity, [3H]SPH was used in the assay instead of [3H]palmitic acid at a saturating concentration of 3 mol %. At the end of the incubation, lipids were extracted and applied on TLC to separate the labeled Cer formed. Initial velocity studies were performed by varying concentrations of SPH at several fixed concentrations of [3H]palmitic acid. Lineweaver-Burk plots were then generated. Secondary plots were next generated by replotting the slopes and the y intercepts of the lines as a function of 1/[palmitic acid]. A random sequential mechanism follows the equation, v =VmaxAB/(Kia Kb+ Ka B +Kb A + AB), whereA and B represent the concentrations of substrates SPH and palmitic acid, respectively, andKia represents the dissociation constant of the enzyme-A complex. The values of KSPH,Kpalmitic acid, V, andKia can be determined from the slopes andy intercepts of the secondary plots as described (21Cleland W.W. Methods Enzymol. 1997; 63: 103-138Crossref Scopus (1929) Google Scholar). Inhibition studies were performed by varying the concentrations of SPH or palmitic acid in the presence or absence of increasing concentrations of the inhibitor as described (22Fromm H.J. Methods Enzymol. 1979; 63: 467-486Crossref PubMed Scopus (97) Google Scholar). Cells plated in 100-mm culture dishes were labeled with 1 μm [3H]SPH (1 μCi/ml) for different times. Lipids were extracted by the method of Bligh and Dyer and separated on TLC using the solvent ethyl acetate:isooctane:acetic acid (50/50/10; v/v). All experiments were performed two or three times (unless indicated) on the enzyme obtained from the single-band fractions of the MonoS column (15El Bawab S. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). When reducing agents were tested, the enzyme was preincubated with these agents for 2 min prior to the assay. When lipid effects were tested, lipids were dried with the substrate, and the mixture was resuspended with Hepes buffer containing Triton X-100 at a final concentration of 0.2%. All linear regression plots were performed using the Cricket Graph V3 program. Further investigation of the ceramidase purified from rat brain revealed that the enzyme also catalyzes the reverse reaction. At first we studied the substrate requirement for this reverse activity. Fig.1 a shows that the enzyme catalyzes the condensation of SPH and palmitic acid into C16-Cer. The enzyme failed to form Cer when palmitoyl-CoA was used as a substrate, indicating that the enzyme acts through a CoA-independent mechanism. Similar observations were first described by Gatt (12Gatt S. J. Biol. Chem. 1963; 238: 3131-3133Abstract Full Text PDF PubMed Google Scholar) using semi-purified ceramidase, and more recently for phytoceramidase from yeast (13Mao C. Xu R. Bielawska A. Obeid L.M. J. Biol. Chem. 2000; 275: 6876-6884Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and from mouse liver (14Tani M. Okino N. Mori K. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 11229-11234Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), but interestingly, a dihydroceramidase from yeast did not display significant reverse activity (23Mao C. Xu R. Bielawska A. Szulc Z.M. Obeid L.M. J. Biol. Chem. 2000; 275: 31369-31378Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Furthermore, fumonisin B1 (FB1), an anti-fungal, is known to inhibit the CoA-dependent Cer synthase activity (24Wang E. Norred W.P. Bacon C.W. Riley R.T. Merill Jr., A.H. J. Biol. Chem. 1991; 266: 14486-14490Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.1 b, FB1 inhibited the CoA-dependent ceramide synthase activity of rat brain microsomes but failed to inhibit the reverse CDase activity purified from rat brain. These results clearly indicate that the two activities represent different enzymes and further attest to the specificity of fumonisin B1. To study the enzyme, we developed an assay with the purified protein as described under “Experimental Procedures.” In this assay, product is separated by a basic Dole extraction, and the activity is linear with time and protein up to 100 μg in the assay (not shown). Having in our hands a reliable assay, we next characterized and investigated the biochemistry of this enzyme activity to gain insight into its physiological role. The purified enzyme showed reverse activity in a narrow pH spectrum (Fig. 2), distinct from the ceramidase activity, which showed a broad pH range from 5.5 to 10 (15). There was very low activity in the alkaline range (pH > 8) and in the acidic range (pH < 5), and the optimum activity was observed at pH 6.5–7. These results indicate that the ceramidase activity is neutral/alkaline whereas the reverse activity is strictly neutral. The addition of MgCl2, MnCl2, CaCl2, and LiCl was without any effect on the reverse activity (Fig.3 a). ZnCl2 and CuCl2 inhibited the enzyme, and total inhibition was observed at around 1 mm. In addition, EDTA up to 10 mm did not show any effect on the reverse activity. These results clearly indicate that the enzyme is totally independent of cations for stimulation of activity. We had shown that reducing agents dithiothreitol and β-mercaptoethanol inhibited ceramidase activity. When tested on the reverse activity, similar effects were observed (Fig. 3 b). Also, ATP up to 10 mm did not affect the activity (not shown). First we studied the specificity for SPH. Sphingosine harbors two chiral centers and therefore exhibits four stereoisomers, only one of which, thed-erythro (2S,3R) is known to exist naturally. As shown in Fig.4 a, the enzyme showed Michaelis-Menten kinetics when d-erythro-SPH was used as substrate. There was very low activity whend-threo, l-threo, orl-erythro-SPH isomers were used, showing a high specificity for the naturally occurring substrate. In addition, there was very low activity withd-erythro-dihydrosphingosine, a sphingolipid metabolite in the de novo pathway (5Michel C. Van Echten-Deckert G. Rother J. Sandhoff K. Wang E. Merrill A.H. J. Biol. Chem. 1997; 272: 22432-22437Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), which differs from d-erythro-SPH in lacking the 4–5trans double bond. Next, we studied the effect of the fatty acid chain. Fig. 4 b shows the kinetic curves when various fatty acids were used, and Table Irepresents the apparent Km andVmax values deduced from the double-reciprocal plots of each fatty acid. The Km values for the fatty acids were all comparable and within a range of 1.1 to 2.2 mol %, but the Vmax values were more variable, ranging from 0.08 to 0.63 μmol/min/mg. Thus, as judged by theVmax/Km ratio, the enzyme showed the highest synthesis rate with myristic acid (highestVmax/Km ratio).Table IApparent Km and Vmax values of various fatty acidsFatty acidKm(app)Vmax(app)Vmax(app)/Km(app)mol %μmol/min/mgMyristate (14:0)1.70.630.37Palmitate (16:0)2.20.470.21Oleate (18:1)1.10.280.24Nervonate (24:1)1.50.250.16Lignocerate (24:0)2.00.080.04The results are obtained from the Lineweaver-Burk plots of the data in Fig. 4 b. Open table in a new tab The results are obtained from the Lineweaver-Burk plots of the data in Fig. 4 b. To study the kinetic mechanism of the enzyme, the reverse activity was measured as a function of varying concentrations of SPH (0.19–1.56 mol %) at five fixed concentrations of [3H]palmitic acid (0.39–6.2 mol %). The Lineweaver-Burk plots of the data were linear and thus followed Michaelis-Menten kinetics (Fig.5 a). The plots intersected to the left side of the ordinates, indicating a sequential kinetic mechanism (21Cleland W.W. Methods Enzymol. 1997; 63: 103-138Crossref Scopus (1929) Google Scholar). To distinguish between ordered sequential mechanism and random sequential mechanism, inhibition studies were performed (22Fromm H.J. Methods Enzymol. 1979; 63: 467-486Crossref PubMed Scopus (97) Google Scholar). First,l-erythro-SPH was used as a dead-end inhibitor to perform these studies. When the reverse activity was measured as a function of SPH concentrations in the absence or presence ofl-erythro-SPH, double-reciprocal plots of the data reflected typical competitive inhibition (Fig.6 a). On the other hand,l-erythro-SPH showed a noncompetitive inhibition pattern when studied as a function of palmitic acid concentration (Fig.6 b). Next, we screened several compounds to find another inhibitor for the second substrate, palmitic acid. Hexadecanol, palmitic acid methyl ester, palmitaldehyde, and myristaldehyde were among the products tested for inhibition. Both of the aldehyde compounds showed inhibition (∼50% at 2.5–3 mol %). Therefore, myristaldehyde was used for the following experiments. When SPH concentrations were varied, a noncompetitive pattern was observed (Fig.6 c), and when varying the concentrations of palmitic acid, a competitive inhibition was observed (Fig. 6 d). These results indicate that the reverse activity follows a random-sequential mechanism (22Fromm H.J. Methods Enzymol. 1979; 63: 467-486Crossref PubMed Scopus (97) Google Scholar). In this mechanism association and dissociation of both SPH and fatty acid are fast, and there is no obligate order binding of the substrates. Secondary plots were next generated (Fig.5 b), and the kinetic constants obtained from the slopes andy intercepts of these plots are presented in TableII (22Fromm H.J. Methods Enzymol. 1979; 63: 467-486Crossref PubMed Scopus (97) Google Scholar). The Km for SPH was 0.16 mol %, and the Km for palmitic acid was 2.1 mol %. These values were close to the apparentKm values obtained in previous and independent analysis for SPH (0.27 mol %, Fig. 4 a) and for palmitate (2.2 mol %, Table I), suggesting that the binding of the first substrate does not affect the binding of the second substrate.Table IIKinetic parameters of the reverse reactionSubstrateKmVmaxKiamol %μmol/min/mgmol %Sphingosine0.160.30.44Palmitate2.10.3C16-ceramide2-aResults were obtained from Ref. 15.1.34.4The data were obtained from the secondary plots shown in Fig.5 b.2-a Results were obtained from Ref. 15El Bawab S. Bielawska A. Hannun Y.A. J. Biol. Chem. 1999; 274: 27948-27955Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar. Open table in a new tab The data were obtained from the secondary plots shown in Fig.5 b. The effects of various sphingolipids and phospholipids on the reverse activity were investigated. These lipids were added at the indicated mol % concentration with the substrates. Fig. 7 a shows that sphingomyelin inhibited the reverse activity with half-maximal inhibition at around 5 mol %. Cerobrosides were less effective. C16-Cer, the product of the reaction, showed moderate inhibition, with 50% inhibition at 10 mol %. Next we studied the effect of various phospholipids and lysophospholipids (Fig. 7 b). Lysophosphatidic acid was without any effect. At 10 mol %, phosphatidylcholine, phosphatidylserine (PS), phosphatidylglycerol, and lysophosphatidylcholine had moderate inhibition of the activity, with maximum inhibition of around 25–50%. Very interestingly, phosphatidic acid (PA) and cardiolipin (CL) inhibited totally the reverse activity and at lower concentrations (2.5–5 mol %). Because the human isoform was found to be localized to mitochondria (16El Bawab S. Roddy P. Qian T. Bielawska A. Lemasters J.J. Hannun Y.A. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) and because CL is a major lipid of mitochondrial membranes, this effect of CL could be of physiological relevance. Thus, we further investigated the effect of this lipid on the enzyme. Surprisingly, and as shown in Fig. 8 a, CL stimulated ceramidase activity within the same range of concentrations that inhibited the reverse activity, with a 2.5-fold increase at 8–10 mol %. These intriguing observations were further investigated to confirm these results. First, the stimulatory effect on ceramidase activity was independent of the pH of the reaction, because the increase of the ceramidase activity was still observed when the reaction was performed at pH 7 (data not shown). Second, because of the negative charges, CL could interfere with the assay extraction. To answer this, at the end of the incubation, the reaction media were dried, total lipids were applied and separated on TLC, and the Cer band was scraped and counted. Results shown in Fig. 8 b indicate that CL still inhibited the reverse activity and activated the ceramidase activity, indicating that CL did not interfere with lipid extraction. Third, CL could inhibit the reverse activity by acting as a donor of fatty acid. To exclude this possibility, the assay was performed in the presence of [3H]SPH and increasing concentrations of CL. Total lipids were then extracted and applied on TLC, and the Cer band was scraped and counted. There was no formation of Cer under these conditions (not shown), indicating that the enzyme uses only free fatty acid, and that CL was not used as a fatty acid donor in a transacylase reaction. Next, the mechanism of activation and inhibition by CL was investigated. Fig.9 a shows that CL increased theVmax of the ceramidase reaction. When varying the concentration of SPH, a competitive type of inhibition of the reverse activity was observed, and when varying the concentration of palmitic acid, CL showed a noncompetitive type of inhibition (Fig.9 b). Those results disclose specific and different effects of CL on ceramidase activity and the reverse activity.Figure 9Mechanism of CL activation and inhibition. The effect of CL on CDase and reverse CDase activities was determined as a function of varying concentrations of the substrates. a, effect of CL on theVmax of ceramidase activity. The assay was performed using [3H]C16-Cer as described under “Experimental Procedures.” b, effect of CL on the reverse reaction. At the end of the assay, lipids were extracted with chloroform/methanol, dried, and applied on TLC to separate the substrates from the product [3H]Cer. The spotscorresponding to Cers were then scraped, and the radioactivity was quantified using liquid scintillation counting. When studied as a function of SPH concentrations, CL decreased the km value, and when studied as a function of palmitic acid concentrations, CL decreased theVmax.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Double-labeling experiments were performed to study the direction of the reaction in the presence of all substrates, and this, in the absence or presence of increasing concentrations of CL. Each substrate, [3H]C16-Cer, SPH, and [14C]palmitic acid, was added at itsKm value (1.3 mol % for Cer, 0.16 mol % for SPH, and 2.2 mol % for palmitic acid), Km/3 or 3 ×Km. As shown in Fig.10 (a and b), the enzyme catalyzed both activities, ceramidase (monitored by the release of [3H]palmitic acid) and reverse CDase activity (monitored by the formation of [14C]Cer). In addition, both activities followed saturation curves, and their ratio was close to unity (Fig. 10 c). In the presence of increasing concentrations of CL, as observed before, CDase activity was stimulated while the reverse activity was inhibited, and this" @default.
• W2110361141 created "2016-06-24" @default.
• W2110361141 creator A5012553792 @default.
• W2110361141 creator A5028663814 @default.
• W2110361141 creator A5038053050 @default.
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• W2110361141 date "2001-05-01" @default.
• W2110361141 modified "2023-09-27" @default.
• W2110361141 title "Biochemical Characterization of the Reverse Activity of Rat Brain Ceramidase" @default.
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