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• W2037892813 abstract "Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229–11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that theN-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside. Previously, we reported two types of neutral ceramidase in mice, one solubilized by freeze-thawing and one not. The former was purified as a 94-kDa protein from mouse liver, and cloned (Tani, M., Okino, N., Mori, K., Tanigawa, T., Izu, H., and Ito, M. (2000) J. Biol. Chem. 275, 11229–11234). In this paper, we describe the purification, molecular cloning, and subcellular distribution of a 112-kDa membrane-bound neutral ceramidase of rat kidney, which was completely insoluble by freeze-thawing. The open reading frame of the enzyme encoded a polypeptide of 761 amino acids having nine putative N-glycosylation sites and one possible transmembrane domain. In the ceramidase overexpressing HEK293 cells, 133-kDa (Golgi-form) and 113-kDa (endoplasmic reticulum-form) Myc-tagged ceramidases were detected, whereas these two proteins were converted to a 87-kDa protein concomitantly with loss of activity when expressed in the presence of tunicamycin, indicating that theN-glycosylation process is indispensable for the expression of the enzyme activity. Immunohistochemical analysis clearly showed that the ceramidase was mainly localized at the apical membrane of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in liver the enzyme was distributed with endosome-like organelles in hepatocytes. Interestingly, the kidney ceramidase was found to be enriched in the raft microdomains with cholesterol and GM1 ganglioside. ceramide 5-bromo-4-chloro-3-indolyl phosphate ceramidase 4-nitrobenzo-2-oxa-1,3-diazole nitro blue tetrazolium polymerase chain reaction sphingosine sphingosine 1-phosphate fluorescein isothiocyanate high performance liquid chromatography polyacrylamide gel electrophoresis phosphate-buffered saline Galβ1–3GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ1–1′Cer Over the past decade, sphingolipids and their metabolites have emerged as a new class of lipid biomodulators of various cell functions (1Hakomori S. Igarashi Y. J. Biochem. (Tokyo). 1995; 118: 1091-1103Crossref PubMed Scopus (369) Google Scholar, 2Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (650) Google Scholar). Ceramide (N-acylsphingosine; Cer),1 a common lipid backbone of sphingolipids, functions as a second messenger in a variety of cellular events including apoptosis and cell differentiation (3Obeid L.M. Linardic C.M. Hannun Y.A. Science. 1993; 256: 1769-1771Crossref Scopus (1618) Google Scholar, 4Okazaki T. Bielawska A. Bell R.M. Hannun Y.A. J. Biol. Chem. 1990; 265: 3125-3128Abstract Full Text PDF Google Scholar). Sphingosine (Sph) has bifunctional effects on cell growth,i.e. it exerts mitogenic (5Zhang H. Buckley N.E. Gibson K. Spiegel S. J. Biol. Chem. 1990; 265: 76-81Abstract Full Text PDF PubMed Google Scholar) and apoptosis inducing (6Ohta H. Sweeney E.A. Masamune A. Yatomi Y. Hakomori S. Igarashi Y. Cancer Res. 1995; 55: 691-697PubMed Google Scholar) activities, depending on the cell type and cell cycle. Sph-1-phosphate (S1P) was found to function as an intra- and intercellular second messenger to regulate cell growth (7Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (818) Google Scholar), motility (8Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y.R. Nature. 2000; 406: 192-195Crossref PubMed Scopus (347) Google Scholar), and morphology (9Van Brocklyn J.R. Tu Z. Edsall L.C. Schmidt R.R. Spiegel S. J. Biol. Chem. 1999; 274: 4626-4632Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Interestingly, S1P inhibits the apoptosis induced by Cer and Fas ligand (10Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind J.S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1357) Google Scholar), indicating that the balance of Cer/Sph/S1P affects cell phenotype. Ceramidase (CDase, EC 3.5.1.23) is an enzyme that catalyzes hydrolysis of the N-acyl linkage of Cer to produce Sph, which can be phosphorylated to S1P by sphingosine kinase (11Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Sph is not produced by de novo synthesis (12Michel C. van Echten-Deckert G. Rother J. Sandhoff K. Wang E. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 22432-22437Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), and thus the activity of CDase is crucial not only for switching off the Cer-induced signaling but also for generation of Sph and S1P. CDase is classified into two categories: acid and neutral/alkaline enzymes depending on pH optimum. Acid CDase is thought to be a housekeeping enzyme to catabolize Cer in lysosomes. The enzyme was purified from human urine (13Bernardo K. Hurwitz R. Zenk T. Desnick R.J. Ferlinz K. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1995; 270: 11098-11102Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and cDNA encoding the enzyme was isolated from cDNA libraries of human (14Koch J. Gartner S. Li C.M. Quintern L.E. Bernardo K. Levran O. Schnable D. Desnick R.J. Schuchman E.H. Sandhoff K. J. Biol. Chem. 1996; 271: 33110-33115Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) and mouse (15Li C.-M. Hong S.-B. Kopal G. He X. Linke T. Hou W.-C. Koch J. Gatt S. Sandhoff K. Schuchman E.H. Genomics. 1998; 50: 267-274Crossref PubMed Scopus (96) Google Scholar). A deficiency of the enzyme could cause Farber disease in which Cer is accumulated in lysosomes (16Sugita M. Dulaney J.T. Moser H.W. Science. 1972; 178: 1100-1102Crossref PubMed Scopus (155) Google Scholar). Neutral/alkaline CDase seems to change the balance of Cer/Sph/S1P in response to various stimuli including cytokines and growth factors, and could modulate the sphingolipid-mediated signaling. For example, the activity of membrane-associated neutral CDase was shown to be up-regulated by platelet-derived growth factor in rat glomerular mesangial cells (17Coroneos E. Martinez M. McKenna S. Kester M. J. Biol. Chem. 1995; 270: 23305-23309Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and the enzyme activity was modulated in a bimodal manner by interleukin-1β in rat hepatocytes (18Nikolva-Karakashian M. Morgan E.T. Alexander C. Liotta D.C. Merrill Jr., A.H. J. Biol. Chem. 1997; 272: 18718-18724Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), resulting in a decrease of Cer concomitantly with an increase of Sph. However, the biological function of the enzyme is still not clear. Recently, cDNAs encoding sphingomyelinase, Sph kinase (19Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), and S1P receptors (Edg family) (20Lee M.J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (890) Google Scholar) have been successively cloned. The functions of sphingolipids are now open for elucidation at the molecular level. In the past few years, molecular cloning of neutral/alkaline CDases, one of the missing links of sphingolipid signaling, has been performed in mice (21Tani 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), human (22Bawab S.E. Roddy P. Qian T. Blielawska A. Lemasters J.J. Hannun Y.H. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), bacteria (23Okino N. Ichinose S. Omori A. Imayama S. Nakamura T. Ito M. J. Biol. Chem. 1999; 274: 36616-36622Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and yeast (24Mao C. Xu R. Bielawska A. Obeid L.M. J. Biol. Chem. 2000; 275: 6876-6884Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). In mice, we found two types of neutral CDase, one solubilized by freeze-thawing and the other not. The former was purified as a 94-kDa protein from mouse liver (25Tani M. Okino N. Mitsutake M. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 3462-3468Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and the cDNA encoding the enzyme was cloned (21Tani 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). In the present paper, we report the purification, characterization, and cDNA cloning of a 112-kDa membrane-bound CDase of rat kidney, which was absolutely resistant to extraction with freeze-thawing and had an optimum pH of 6–7. It is worth noting that neutral/alkaline CDase of human brain is specifically localized in mitochondria, suggesting the existence of a Cer pool in this organelle (22Bawab S.E. Roddy P. Qian T. Blielawska A. Lemasters J.J. Hannun Y.H. J. Biol. Chem. 2000; 275: 21508-21513Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). On the other hand, we show here using a specific antibody against the neutral CDase that the enzyme was mainly localized at apical membranes of proximal tubules, distal tubules, and collecting ducts in rat kidney, while in rat liver the enzyme was distributed with endosome-like organelles in hepatocytes. Furthermore, the kidney CDase was recovered in the detergent-insoluble, cholesterol, and GM1-enriched fractions by sucrose density gradient centrifugation, suggesting that the enzyme is present in the raft microdomains. Monoclonal antibody YA30 against LGP85 (Limp-2) and CHOP cells were kind gifts from Dr. K. Akasaki (Fukuyama University, Japan) and Dr. K. Nara (Mitsubishi Kasei Institute of Life Sciences, Japan), respectively. Horseradish peroxidase-labeled anti-mouse IgG and anti-rabbit IgG were purchased from EY Laboratories and Santa Cruz Biotechnology, respectively. DEAE-Sepharose FF, phenyl-Sepharose 6FF, chelating Sepharose FF, HiTrap lentil lectin, HiTrap ConA, HiLoad 16/60 Superdex 200 pg, Percoll, ECL plus, FITC-labeled anti-mouse Ig antibody, and Cy3-labeled anti-rabbit IgG antibody were from Amersham Pharmacia Biotech. Precoated Silica Gel 60 TLC plates were obtained from Merk (Germany). Amplex Red (N-acetyl-3,7-dihydroxyphenoxazine) and cholesterol oxidase were from Molecular Probe and Toyobo Co. (Japan), respectively. Various [14C]Cers and C12-NBD-Cer were prepared as described in Ref. 26Ito M. Mitsutake S. Tani M. Kita K. Methods Enzymol. 1999; 311: 682-689Crossref Scopus (12) Google Scholar. HEK293 cell (JCRB9068, established by F. L. Graham) was from the Human Science Research Resource Bank. All other reagents were of the highest purity available. CDase activity was measured using C12-NBD-Cer as a substrate (25Tani M. Okino N. Mitsutake M. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 3462-3468Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, 550 pmol of C12-NBD-Cer was incubated at 37 °C for 30 min with an appropriate amount of the enzyme in 20 µl of 25 mm Tris-HCl buffer, pH 7.5, containing 0.25% Triton X-100. The reaction was stopped by heating in a boiling water bath for 5 min. After being dried up with a Speed Vac concentrator (Savant Instruments, Inc.), the sample was dissolved in 30 µl of chloroform/methanol (2/1, v/v), and applied to a TLC plate, which was developed with chloroform, methanol, 25% ammonia (90/20/0.5, v/v). The NBD-dodecanoic acid released and C12-NBD-Cer produced were quantified with a Shimadzu CS-9300 chromatoscanner (Shimadzu, Japan). One enzyme unit was defined as the amount capable of catalyzing the release of 1 µmol of NBD-dodecanoic acid/min from C12-NBD-Cer under the conditions described above. A value of 10−3 and 10−6 units of enzyme was expressed as 1 milliunit and 1 microunit, respectively. To characterize the CDase, various cations at 5 mm as a final concentration were added, or 150 mm GTA buffer at different pH values was used instead of Tris-HCl buffer. In some cases, 100 pmol of [14C]Cer (C16:0/d18:1) was used instead of the fluorescent Cer as a substrate. The reverse hydrolysis reaction of the CDase was determined by the method described in Ref. 25Tani M. Okino N. Mitsutake M. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 3462-3468Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar. Fresh rat kidneys (141 g wet weight from 53 rats) were homogenized with Polytron-RT3000 in 300 ml of 0.25 m sucrose containing 1 mmEDTA. The homogenate was centrifuged at 600 × g for 5 min to remove debris. The supernatant was centrifuged at 27,000 ×g for 30 min and the pellet obtained (membrane fraction) was re-suspended in 200 ml of 10 mm potassium phosphate buffer, pH 7.0, containing 1% Triton X-100 and 1% Tween 20. After sonication for 2 min on ice, the homogenate was then centrifuged at 105,000 × g for 90 min. The supernatant was used as a detergent-extracted fraction. The membrane fractions of rat kidney and liver were suspended in 100 µl of 0.25 msucrose containing 1 mm EDTA, 1 mmphenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin. The solutions were frozen in ethanol-dry ice immediately and thawed in warm water at 37 °C. The procedure was repeated the number of times indicated. After centrifugation at 105,000 × g for 60 min, the supernatant obtained was used as the soluble fraction. The precipitate was suspended in 100 µl of 20 mm Tris-HCl buffer, pH 7.5, containing 0.2% Triton X-100 and used as a membrane-bound fraction. The CDase (1.14 units), extracted from the membrane fraction of rat kidney with a mixture of 1% Triton X-100 and 1% Tween 20, was loaded onto a DEAE-Sepharose FF column (200 ml) equilibrated with 20 mm Tris-HCl buffer, pH 7.5, containing 0.1% Lubrol PX (buffer A) at a flow rate of 5 ml/min using a BPLC-600FC HPLC system (Yamazen Co., Osaka, Japan). After sample loading, the column was washed with 300 ml of buffer A followed by a linear gradient of 0–1 m NaCl in buffer A at a flow rate of 5 ml/min. The eluted fractions containing the CDase activity were pooled, and then applied to a phenyl-Sepharose 6FF column (100 ml) at a flow rate of 5 ml/min using a BPLC-600FC HPLC system. The column was washed with 200 ml of 20 mm Tris-HCl buffer, pH 7.5, and then the CDase was eluted using a linear gradient of 0–2% Lubrol PX in 20 mm Tris-HCl buffer, pH 7.5, at a flow rate of 5 ml/min. The eluate was pooled and loaded onto a column of chelating Sepharose FF (50 ml) at a flow rate of 5 ml/min using a BPLC-600FC HPLC system. The column was washed with 200 ml of buffer A and 100 ml of 20 mm Tris-HCl buffer, pH 7.5, containing 0.5 mNaCl and the CDase activity was eluted with 20 mm Tris-HCl buffer, pH 7.5, containing 2 m NH4Cl at a flow rate of 5 ml/min. The fractions containing the enzyme activity were pooled and concentrated about 10-fold using a MiniTan ultrafiltration system (Millipore). The buffer was exchanged to 20 mmTris-HCl buffer, pH 7.5, using the same apparatus. The enzyme solution was loaded onto a HiTrap lentil lectin column (1 ml, Amersham Pharmacia Biotech) equilibrated with 20 mm Tris-HCl buffer, pH 7.5, containing 1 mm MnCl2, 1 mmCaCl2, and 0.5 m NaCl at a flow rate of 0.4 ml/min using a BioCAD system (Applied Biosystems). The CDase was eluted with 20 mm Tris-HCl buffer, pH 7.5, containing 0.5m methyl-α-d-glucoside. The active fractions were pooled and concentrated using a Centriprep (Millipore) and loaded onto a HiLoad 16/60 Superdex 200 pg column (Amersham Pharmacia Biotech) equilibrated with 20 mm Tris-HCl buffer, pH 7.5, containing 0.15 m NaCl and 0.3% Lubrol PX at a flow rate of 0.8 ml/min using a BioCAD system. The purified CDase was concentrated with a Y-shaped gel a modified form of a funnel-shaped gel (27Hatt P.D. Quadroni M. Staudenmann W. James P. Eur. J. Biochem. 1997; 246: 336-343Crossref PubMed Scopus (23) Google Scholar). After the concentration, the 112-kDa protein band localized with Coomassie Brilliant Blue was cut out, equilibrated with SDS sample buffer, and loaded again on a 7.5% SDS-PAGE gel. After electrophoresis, the gel was blotted on a polyvinylidine difluoride membrane and stained with Coomassie Brilliant Blue. The 112-kDa protein (about 3 µg) was cut out and treated in situ with lysylendopeptidase AP-1 (Wako Pure Chemical Industries, Osaka, Japan). Peptides released from the membrane were fractionated by reversed-phase HPLC using a C8 column (1.0 × 100 mm), and sequenced using a pulse-liquid phase protein sequencer (Procise cLc, Applied Biosystems). The sequences of four peptides obtained after digestion with lysylendopeptidase AP-1 showed high identity to the mouse liver neutral CDase (21Tani 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), and thus we designed two primers based on the nucleotide sequence of the mouse enzyme. PCR using sense (5′-AGGAAATGTTGCTAATGTGC-3′) and antisense primers (5′-GGTGACACGTCTCCGAGAT-3′) was performed with the cDNA library of rat kidney (Takara Shuzo Co., Otsu, Japan) as a template in a GeneAmp PCR System 9700 (Applied in Biosystems) using AmpliTaq Gold (Applied in Biosystems). The cycling parameters for PCR were 94 °C for 30 s, 51 °C for 30 s, and 72 °C for 30 s, and the cycle number was 40. After this amplification, a 325-base pair PCR product containing the sequence of rat CDase was obtained. To obtain the full-length cDNA encoding the rat CDase, colony hybridization was performed using the α-32P-labeled 325-base pair PCR product as a probe after concentration of the CDase cDNA with a CloneCaptureTM Selection Kit (CLONTECH). The probe was labeled with [α-32P]dCTP using Ready-To-Go DNA labeling kit (Amersham Pharmacia Biotech). Colony hybridization was carried out by the standard method (28Sambrook H. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Nucleotide sequences were determined by the dideoxynucleotide chain termination method with a Bigdye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), and a DNA Sequencer (model 377A, Applied Biosystem). A cDNA fragment encoding the open reading frame of rat CDase was prepared by PCR using 5′ primer containing aHindIII site (5′-AGTAAGCTTATCGAAAACCACAAAGATTCAGGGA-3′) and 3′ primer containing a XhoI site (5′-GCCGCTCGAGAGTAGTGACAATTTCAAAAGGGGAAGA-3′) and the cloned rat cDNA (pAPkCD) as a template. The PCR product was inserted into theHindIII and XhoI sites of pET23b vector (Novagen) with a COOH-terminal histidine tag. Escherichia coli strain BL21(DE3) was transformed with the construct in the presence of ampicillin (100 µg/ml). To obtain the recombinant CDase, 2 ml of overnight culture was inoculated into 100 ml of LB in the presence of ampicillin and incubated at 37 °C. When the absorbance at 600 nm reached 0.6, isopropyl-1-thio-β-d-galactopyranoside was added to a final concentration of 1 mm, and incubated for no more than 5 h at 37 °C. Cells were harvested by centrifugation at 5,000 × g for 10 min at 4 °C, and the pellet was suspended in 10 mm Tris-HCl buffer, pH 7.5, containing 1% Triton X-100 and 1 mm EDTA. After centrifugation at 15,000 × g for 10 min, the pellet (inclusion bodies) was lysed by sonication in 50 mmTris-HCl buffer, pH 7.5, containing 8 m urea. Recombinant protein was purified using a HiTrap chelating column (Ni2+) according to the manufacturer's instructions. Purified protein was dialyzed against distilled water before being used for immunization. From a rabbit immunized with the purified recombinant CDase, antiserum was obtained and purified with a HiTrap Protein A column according to the manufacturer's instructions. CHOP cells, Chinese hamster ovary cells that express polyoma LT antigen for supporting efficient replication of eukaryotic expression vectors (29Hefferman M. Dennis W.J. Nucleic Acids Res. 1991; 19: 85-92Crossref PubMed Scopus (94) Google Scholar), were grown in a α-minimal essential medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin in a humidified incubator containing 5% CO2. HEK293 cells, human embryonic kidney cell, were grown in a Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 60 µg/ml kanamycin in a humidified incubator containing 5% CO2. cDNA transfection was carried out using LipofectAMINETMPlus (Life Technologies, Inc.) according to the instructions of the manufacturer. To obtain Myc-tagged CDase, cDNA encoding the CDase was subcloned into pcDNA3.1/Myc-His(+) vector (Invitrogen Co.) by PCR using a 5′ primer with a KpnI restriction site (5′-AGGGTACCGAAATGGCAAAGCGAACCTTCTCC-3′) and a 3′ primer with aXhoI restriction site and disrupted stop codon (5′-GCCGCTCGAGAGTAGTGACAACTTCAAAAGGAGAAGA-3′). Cells were treated with tunicamycin to block the N-glycosylation of neutral CDase. Medium containing tunicamycin (10 µg/ml) was added at 4 h after transfection of CDase gene and cells were harvested after 12 h. Measurement of protein was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as standard. SDS-PAGE was carried out according to the method of Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Protein transfer onto a polyvinyldifluoride membrane was performed using TransBlot SD (Bio-Rad) according to the method described in Ref. 31Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar. After treatment with 3% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (T-TBS) for 1 h, the membrane was incubated with first antibody (anti-neutral CDase antibody, anti-Myc antibody (Invitrogen), or anti-CD71 antibody (Harian Sera-Lab)) for 1 day at 4 °C. After a wash with T-TBS, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 2 h. After another wash with T-TBS, the ECL reaction was performed for 2–3 min as recommended by the manufacturer, and chemiluminescent signals were visualized on STORM (Amersham Pharmacia Biotech). A Percoll density gradient centrifugation was performed according to the method described in Ref.32Symons L.J. Jonas A.J. Anal. Biochem. 1987; 164: 382-390Crossref PubMed Scopus (19) Google Scholar. The tissues were homogenized for 15–20 strokes in 20 mm Tris-HCl buffer, pH 7.0, containing 0.25 msucrose and 1 mm EDTA with a glass-Teflon homogenizer. The homogenate was centrifuged at 750 × g for 10 min. The supernatant obtained was centrifuged at 20,000 × g for 10 min, and the pellet was resuspended in 20 mm Hepes-KOH buffer, pH 7.0, containing 0.25 m sucrose. This suspension was mixed with isotonic Percoll solution (1 ml of 200 mmHepes-KOH, pH 7.0, containing 0.25 m sucrose was mixed with 9 ml of Percoll) at a ratio of 55/45 (v/v) and then centrifuged at 35,000 × g for 90 min using a 10-ml centrifuge tube. The resulting gradient was divided into 10 fractions from top to bottom. Activities of β-galactosidase and alkaline phosphatase were measured using chlorophenol red β-d-galactopyranoside (Roche Molecular Biochemicals) and NBT/BCIP (Roche Molecular Biochemicals) as substrates, respectively. An appropriate amount of each fraction was incubated with 50 µl of 5 mmchlorophenol red β-d-galactopyranoside containing 2.5 mm MgCl2 or NBT/BCIP (3.75/1.88 µg) in 100 µl of 100 mm Tris-HCl buffer, pH 9.5, containing 100 mm NaCl and 5 mm MgCl2 at 37 °C for a given period. After incubation, absorbance at 574 and 550 nm were measured for quantification of β-galactosidase and alkaline phosphatase activities, respectively. Samples (rat kidney and liver) were fixed with 4% paraformaldehyde in PBS overnight at 4 °C, rinsed with PBS and 50 mmNH4Cl in PBS, and then infiltrated with 20% sucrose in PBS overnight at 4 °C. The materials were embedded in OCT compound, rapidly frozen using liquid nitrogen, and stored at −80 °C. The frozen materials were cut into 8-µm thick sections using a cryostat (Leica CM1850) and mounted on poly-l-lysine-coated glass slides. After treatment with 5% skim milk in PBS (blocking buffer) for 20 min at room temperature, the samples were incubated with the anti-neutral CDase antibody diluted 1:100 with a blocking buffer for 2 h at room temperature followed by incubation with Cy3-labeled anti-rabbit IgG (Amersham Pharmacia Biotech) at room temperature for 1 h. For controls, the primary antibody was replaced by preimmune serum IgG. For double labeling, the samples were stained with anti-neutral CDase and anti-LGP85 followed by incubation with a mixture of Cy3-labeled anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ig (Amersham Pharmacia Biotech). Immunostained samples were examined with a confocal laser scanning microscope (Olympus LSM-GB200). A part of immunostained samples were incubated with FITC-conjugated phalloidin (Sigma) to visualize actin filaments. A Northern blot membrane loaded with ∼2 µg of poly(A)+ RNA per lane from 8 different rat tissues (CLONTECH) was hybridized with a 2.0-kilobase EcoRI fragment of pAPkCD, which was gel-purified and labeled with [α-32P]dCTP using the Multiprime DNA labeling system (Amersham Pharmacia Biotech). Hybridization was carried out at 42 °C for 20 h. Detection and quantification were performed using a BAS 1500 imaging analyzer (Fuji Film, Tokyo, Japan). A detergent-insoluble lipid raft was prepared as described in Refs. 33Hansen G.H. Niels-Christiansen L.L. Thorsen E. Immerdal L. Danielsen M. J. Biol. Chem. 2000; 275: 5136-5142Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholarand 34Roper K. Corbeil D. Huttner W.B. Nat. Cell Biol. 2000; 2: 582-592Crossref PubMed Scopus (483) Google Scholar with some modification. In brief, a fresh rat kidney (200 mg wet weight) was homogenized in 5 ml of 50 mm Tris-HCl buffer, pH 7.5, containing 150 mm NaCl, 2 µg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride (homogenized buffer) with a manually operated Teflon-glass homogenizer (20 strokes). The homogenate was centrifugation at 500 × g for 5 min, and the resulting supernatant was then centrifuged at 48,000 ×g for 30 min to obtain a pellet of total membrane. The pellet was resuspended in 1.5 ml of the homogenized buffer containing 0.5% Lubrol 17A17 (serva), extracted for 30 min on ice, and mixed with an equal volume of 80% sucrose in the same buffer. The raft fraction was prepared by layering 5–30% sucrose on top of the extract, followed by centrifugation at 200,000 × g for 20 h. After centrifugation, 10 fractions were collected from top to bottom of the gradient and dialyzed against distilled water. The free-cholesterol in each fraction was measured using a cholesterol oxidase/peroxidase (35Gamble W. Vaughan M. Kruth H.S. Avigan J. J. Lipid Res. 1978; 19: 1068-1070Abstract Full Text PDF PubMed Google Scholar), withN-acetyl-3,7-dihydroxyphenoxazine (Amplex Red, Molecular Probe) as substrate instead of p-hydroxyphenylacetic acid. Detection of ganglioside GM1 was performed using a biotin-labeled cholera toxin B subunit as described below. An aliquot of 50 µl of each fraction was dried up, dissolved in 10 µl of chloroform/methanol (2/1, v/v), and applied to a Polygram Silica G TLC plate (Macherey-Nagel, Germany). After being developed with chloroform, methanol, 0.02% CaCl2 (5/5/1, v/v), the TLC plate was blocked with 1% skim milk-TBS for 1 h, and incubated with biotin-labeled cholera toxin B subunit (1 µg/ml in T-TBS) for 2 h and then streptavidin-alkaline phosphatase (Sigma) for 1 h at room temperature. Detection of alkaline phosphatase was conducted using NBT/BCIP as a substrate as described above. As shown in Fig. 1, the CDase of rat liver was solubilized from the membrane fraction by freeze-thawing (A), whereas the enzyme of rat kidney was completely resistant to extraction with freeze-thawing (B). This discrepancy was also observed when the neutral CDase was extracted from mouse liver and kidney (25Tani M. Okino N. Mitsutake M. Tanigawa T. Izu H. Ito M. J. Biol. Chem. 2000; 275: 3462-3468Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). The neutral CDase of mouse liver was easily solubilized by freeze-thawing, and purified as a 94-kDa protein, and thus cDNA encoding the enzyme was cloned (21Tani 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 (9" @default.
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• W2037892813 title "Purification, Characterization, Molecular Cloning, and Subcellular Distribution of Neutral Ceramidase of Rat Kidney" @default.
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