Namalva (or Namalwa) interferon (IFN)-alpha was partially purified using a combination of conventional methods and modified acid-ethanol extraction. Four mouse monoclonal antibodies against Namalva IFN-alpha were prepared by hybridoma technology after immunization with Namalva IFN-alpha thus purified. Three of these monoclonal antibodies recognized the same or a similar epitope on Namalva IFN-alpha. One of these antibodies was paired with the fourth recognizing a different epitope and used respectively as enzyme-conjugated antibody and solid-phase antibody in our one step enzyme immunoassay (EIA) for IFN-alpha. This assay is simple and was able to detect as little as 5 pg of IFN-alpha in 100 microliters of sample in the short time of 5 hr. There was a good correlation between the EIA and bioassay. The use of one of the monoclonal antibodies as an immunoadsorbant to purify Namalva IFN-alpha is also described.
We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (P o), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity withP o ∼1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity. We characterized type 3 ryanodine receptor (RyR3) purified from rabbit diaphragm by immunoaffinity chromatography using a specific antibody. The purified receptor was free from 12-kDa FK506-binding protein, although it retained the ability to bind 12-kDa FK506-binding protein. Negatively stained images of RyR3 show a characteristic rectangular structure that was indistinguishable from RyR1. The location of the D2 segment, which exists uniquely in the RyR1 isoform, was determined as the region around domain 9 close to the corner of the square-shaped assembly, with use of D2-directed antibody as a probe. The RyR3 homotetramer had a single class of high affinity [3H]ryanodine-binding sites with a stoichiometry of 1 mol/mol. In planar lipid bilayers, RyR3 displayed cation channel activity that was modulated by several ligands including Ca2+, Mg2+, caffeine, and ATP, which is consistent with [3H]ryanodine binding activity. RyR3 showed a slightly larger unit conductance and a longer mean open time than RyR1. Whereas RyR1 showed two classes of channel activity with distinct open probabilities (P o), RyR3 displayed a homogeneous and steeply Ca2+-dependent activity withP o ∼1. RyR3 was more steeply affected in the channel activity by sulfhydryl-oxidizing and -reducing reagents than RyR1, suggesting that the channel activity of RyR3 may be transformed more precipitously by the redox state. This is also a likely explanation for the difference in the Ca2+ dependence of RyR3 between [3H]ryanodine binding and channel activity. Intracellular Ca2+ stores play a critical role in regulation of the cytosolic Ca2+ concentration in various cells. Ryanodine receptors (RyRs) 1The abbreviations used are: RyR, ryanodine receptor; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid; CICR, Ca2+-induced Ca2+ release; DTT, dithiothreitol; FKBP12, 12-kDa FK506-binding protein; GST, glutathioneS-transferase; MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SR, sarcoplasmic reticulum; PVDF, polyvinylidene difluoride; AMP-PCP, adenosine 5′-(β,γ-methylenetriphosphate); pCMPS, p-chloromercuriphenylsulfonic acid belong to a family of Ca2+ release channels of the Ca2+ stores (1Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (445) Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar, 4Sorrentino V. Adv. Pharmacol. 1995; 33: 67-90Crossref PubMed Scopus (72) Google Scholar, 5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 6Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar, 7Wagenknecht T. Radermacher M. Curr. Opin. Struct. Biol. 1997; 7: 258-265Crossref PubMed Scopus (53) Google Scholar). To date, three isoforms of RyR that are encoded by distinct genes have been identified in mammalian tissues. Type 1 RyR (RyR1) is primarily expressed in skeletal muscles and plays a crucial role in excitation-contraction (E-C) coupling, a process whereby depolarization in the sarcolemma triggers Ca2+ release from sarcoplasmic reticulum (SR) resulting in muscle contraction (6Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar, 8Rios E. Pizarro G. Stefani E. Annu. Rev. Physiol. 1992; 54: 109-133Crossref PubMed Scopus (132) Google Scholar, 9Schneider M.F. Annu. Rev. Physiol. 1994; 56: 463-484Crossref PubMed Scopus (258) Google Scholar). Type 2 RyR (RyR2) is the primary isoform in heart and is involved in the E-C coupling in cardiac muscle. cDNA and mRNA for type 3 RyR (RyR3) was first identified in mink lung epithelial cells and in specific regions (hippocampus, thalamus, and corpus striatum) of rabbit brain (3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar, 4Sorrentino V. Adv. Pharmacol. 1995; 33: 67-90Crossref PubMed Scopus (72) Google Scholar). Recent studies revealed that mRNAs for all these isoforms can be widely detected in various tissues, suggesting potential coexpression of multiple isoforms (5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar). RyR1 and RyR2 have been purified from skeletal and cardiac muscles, respectively, and are well characterized (1Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (445) Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar, 5Sutko J.L. Airey J.A. Physiol. Rev. 1996; 76: 1027-1071Crossref PubMed Scopus (365) Google Scholar, 7Wagenknecht T. Radermacher M. Curr. Opin. Struct. Biol. 1997; 7: 258-265Crossref PubMed Scopus (53) Google Scholar). A homotetramer of the ∼560-kDa subunit is a functional unit and shows a characteristic structure with 4-fold symmetry that is identical to feet which span the gap between the transverse tubule and terminal cisterna of SR (6Franzini-Armstrong C. Protasi F. Physiol. Rev. 1997; 77: 699-729Crossref PubMed Scopus (596) Google Scholar, 7Wagenknecht T. Radermacher M. Curr. Opin. Struct. Biol. 1997; 7: 258-265Crossref PubMed Scopus (53) Google Scholar). A homotetramer can specifically bind one [3H]ryanodine molecule with a K D of nanomolar order, after which it is named RyR (1Fleischer S. Inui M. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 333-364Crossref PubMed Scopus (445) Google Scholar, 2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar). When incorporated into planar lipid bilayers, the receptor demonstrates cation channel activity with a large conductance showing properties of Ca2+-induced Ca2+ release (CICR) (2Meissner G. Annu. Rev. Physiol. 1994; 56: 485-508Crossref PubMed Scopus (843) Google Scholar, 3Ogawa Y. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 229-274Crossref PubMed Scopus (229) Google Scholar). Knowledge about RyR3, in contrast, is much less than that about RyR1 or RyR2 because of its minuscule amount. An excess amount of other coexisting isoforms of similar biochemical characteristics has prevented us from isolating RyR3 by conventional purification procedures. To overcome this difficulty, we utilized an antibody specific to RyR3 (anti-RyR3) as a tool for isolation of the protein. Frog skeletal muscle expresses two isoforms of RyRs (α- and β-RyR) in almost equal amounts (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). Cloning and sequencing of their cDNAs revealed that α- and β-RyRs are homologues of RyR1 and RyR3, respectively (11Oyamada H. Murayama T. Takagi T. Iino M. Iwabe N. Miyata T. Ogawa Y. Endo M. J. Biol. Chem. 1994; 269: 17206-17214Abstract Full Text PDF PubMed Google Scholar). Taking advantage of the similarity between RyR3 and β-RyR, we have successfully raised and purified anti-RyR3 against a peptide of amino acid sequence 4375–4387 of rabbit RyR3 (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Selective immunoprecipitation with the antibody revealed properties of RyR3 in rabbit brain (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and diaphragm (13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). RyR3 forms a homotetramer of ∼550-kDa subunit, which is slightly smaller than those of RyR1 and RyR2, as is the case for β-RyR. It shows high affinity [3H]ryanodine binding which is activated by micromolar Ca2+, adenine nucleotides, and caffeine, and inhibited by millimolar concentrations of Ca2+ and Mg2+, procaine, and ruthenium red, indicating that it may function as a CICR channel. The contents of RyR3 in rabbit diaphragm and brain were estimated to be less than 1 and 0.06%, respectively, of RyR1 in skeletal muscle (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). In this paper, we describe the structural and functional characterization of RyR3 that was successfully purified from rabbit diaphragm by immunoaffinity chromatography. [3H]Ryanodine binding and single channel recordings of the molecule revealed several unique properties of RyR3. We also defined the location of the differential segment in the assembly of the RyR1 molecule, which could be one of the origins for the difference in function between the two isoforms. During the course of publication of our results, Jeyakumaret al. (14Jeyakumar L.H. Copello J.A. O'Malley A.M. Wu G.M. Grassucci R. Wagenknecht T. Fleischer S. J. Biol. Chem. 1998; 273: 16011-16020Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) independently reported the purification of RyR3. Anti-RyR3 antibody against a peptide corresponding to the amino acid sequence 4375–4387 of rabbit RyR3 (RyR3-peptide) was produced in rabbits and purified by affinity chromatography with frog β-RyR-bound polyvinylidene difluoride (PVDF) membranes (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Monoclonal antibody 3F4 against a 12-kDa FK506-binding protein (FKBP12) and human recombinant FKBP12 (hFKBP12) were produced as described previously (15Kobayashi M. Ohtsuka K. Tamura K. Ohara K. Fujihira S. Hirano Y. Kusunoki C. Hayashi M. Satoh S. Katayama N. Tsutsumi T. Nakamura K. Niwa M. Kohsaka M. Transplant. Proc. 1993; 25: 655-657PubMed Google Scholar, 16Shirakata Y. Kobayashi M. Ohtsuka K. Sugano M. Terajima H. Ikai I. Okajima H. Egawa H. Inomata Y. Inamoto T. Tanaka K. Yamaoka Y. Transplantation. 1995; 60: 1582-1587Crossref PubMed Scopus (13) Google Scholar). Monoclonal antibody XA7 which reacts with RyR1 (17Imagawa T. Smith J.S. Coronado R. Campbell K.P. J. Biol. Chem. 1987; 262: 16636-16643Abstract Full Text PDF PubMed Google Scholar) and 34C which recognizes all the three mammalian RyR isoforms (18Airey J.A. Beck C.F. Murakami K. Tanksley S.J. Deerinck T.J. Ellisman M.H. Sutko J.L. J. Biol. Chem. 1990; 265: 14187-14194Abstract Full Text PDF PubMed Google Scholar) were purchased from Upstate Biotechnology, Inc., and Affinity Bioreagents, Inc., respectively. [3H]Ryanodine (60–90 Ci/mmol) was purchased from NEN Life Science Products. Goat anti-rabbit IgG-agarose and glutathione-agarose were obtained from Sigma. Egg lecithin (egg total phosphatide extract) was from Avanti Polar-Lipids. All other reagents were of analytical grade. Heavy fraction of SR vesicles was prepared from rabbit diaphragm according to Murayama and Ogawa (13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) in the presence of a mixture of protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml antipain, 2 μg/ml pepstatin A, and 2 μg/ml chymostatin). The isolated membrane vesicles were quickly frozen in liquid N2and stored at −80 °C until used. RyR3 was purified by a combination of sucrose density gradient ultracentrifugation and immunoprecipitation. Anti-RyR3 beads were prepared by mixing anti-rabbit IgG-agarose beads with the purified anti-RyR3 antibody, followed by washing with a buffer containing 0.15 m NaCl, 20 mm sodium phosphate, pH 7.2. These anti-RyR3 beads can be stored at 4 °C for 3 months without a significant decrease in the RyR3-binding activity. Rabbit diaphragm SR vesicles (10 mg) were solubilized in 4% CHAPS and 2% egg lecithin in 2.5 ml of 0.5 m NaCl, 20 mmTris-HCl, pH 7.4, and 2 mm dithiothreitol (DTT) including the above protease inhibitors (buffer A) to isolate tetrameric RyRs by ultracentrifugation on 5–20% linear gradients of sucrose (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). Fractions around ∼15% sucrose containing tetrameric RyRs were incubated overnight at 4 °C with 100 μl of the anti-RyR3 beads. The beads were washed five times with buffer A containing 1% CHAPS and 0.5% egg lecithin, and then incubated overnight at 4 °C with 30 μm RyR3- peptide in the same buffer to dissociate RyR3 from the antibody. Finally, the supernatant containing RyR3 was applied to a small gel filtration column (Centri-sep, Applied Biosystems Inc.) to remove a large amount of the RyR3-peptide. RyR1 was purified from rabbit back muscle in which virtually no RyR3 protein was detected (13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) by sucrose gradients and Mono-Q anion exchange column chromatography as described (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). Protein concentrations of the purified RyR1 were determined by the method of Kaplan and Pedersen (19Kaplan R.S. Pedersen P.L. Anal. Biochem. 1985; 150: 97-104Crossref PubMed Scopus (187) Google Scholar) with Amido Black 10B using bovine serum albumin as a standard. The amount of RyR3 was determined by densitometry of its band on Coomassie Brilliant Blue-stained SDS-polyacrylamide gel with a MasterScan densitometer. The purified RyR1 or RyR2 was used as a standard in determinations where it showed a linear relationship up to 100 ng (data not shown). SDS-PAGE was performed on 2–12% linear gradient gel with standards of 205 (in kDa) (myosin heavy chain), 116 (β-galactosidase), 97.4 (phosphorylase b), 66 (bovine serum albumin), 45 (ovalbumin), and 29 (carbonic anhydrase) (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Gels were stained with Coomassie Brilliant Blue. The separated proteins were electrophoretically transferred overnight onto PVDF membranes at 40 V in the presence of 0.02% SDS (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Immunodetection was carried out with an ECL system (Amersham Pharmacia Biotech) using peroxidase-conjugated secondary antibodies. Primary antibodies were diluted as follows: 1:1,000 for anti-RyR3, 1:5,000 for XA7, 1:5,000 for 34C, and 1:1,000 for 3F4. When probed by these antibodies, the same transferred PVDF membrane was repeatedly used. Binding of FKBP12 to RyR3 was determined using glutathione S-transferase (GST)-hFKBP12 fusion protein (GST-FKBP) (20Timerman A.P. Onoue H. Xin H.-B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). The plasmid construction of GST-FKBP was essentially the same as previously reported (21Chen S.R. Zhang L. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11953-11957Crossref PubMed Scopus (53) Google Scholar). A plasmid pFKBP333 for hFKBP12 (15Kobayashi M. Ohtsuka K. Tamura K. Ohara K. Fujihira S. Hirano Y. Kusunoki C. Hayashi M. Satoh S. Katayama N. Tsutsumi T. Nakamura K. Niwa M. Kohsaka M. Transplant. Proc. 1993; 25: 655-657PubMed Google Scholar) was subjected to PCR to placeBamHI and EcoRI restriction sites at the 5′- and 3′-end of hFKBP12, respectively. The set of primers was as follows: 5′-CGGGATCCAGATGGGAGTGCAGGTGGAAAC-3′ (hFKBP12-F) and 5′-CGGAATTCTCATTCCAGTTTTAGAAGCTCC-3′(hFKBP12-R). The PCR product was digested with BamHI and EcoRI restriction enzymes and ligated into the BamHI/EcoRI sites of pGEX-3X vector (Amersham Pharmacia Biotech). Expression and purification of the fusion protein was carried out according to the manufacturer's instructions. The purified GST-FKBP was recognized by the anti-FKBP12 antibody (data not shown). 25 μl of glutathione-agarose beads was incubated for 2 h at room temperature with 10 μg of control GST or GST-FKBP in 0.17m NaCl, 20 mm Tris-HCl, pH 7.4, 0.1% CHAPS, 2 mm DTT, and 0.3 m sucrose (buffer B) with gentle mixing. After washing twice with buffer B, the beads were incubated for 1 h at 37 °C with 1 μg of the purified RyR1 or RyR3 in buffer B. The beads were washed three times with buffer B and proteins bound to the beads were subjected to SDS-PAGE. To determine with high sensitivity the [3H]ryanodine binding activity with a small amount of RyR3 protein, we separated protein-bound ryanodine from the free ligand using a small scale gel filtration column (Centri-sep) in a centrifuge. The purified RyR3 was incubated with 8.5 nm [3H]ryanodine for 5 h at 25 °C in 30–50 μl of a binding buffer containing 0.17 m NaCl, 10 mm MOPSO/NaOH, pH 6.8, 2 mm DTT, 1% CHAPS, 0.5% egg lecithin, 4 mm AMP and various concentrations of Ca2+ buffered with 10 mm EGTA. Free Ca2+ concentrations were calculated using the value of 8.79 × 105m−1 as the apparent binding constant for Ca2+ of EGTA (22Harafuji H. Ogawa Y. J. Biochem. (Tokyo). 1980; 87: 1305-1312Crossref PubMed Scopus (346) Google Scholar). Then, an aliquot of the sample (10–20 μl) was applied to the Centri-sep column that had been equilibrated with 1 m NaCl, 10 mm MOPSO/NaOH, pH 6.8, 1% CHAPS, 0.5% egg lecithin, 2 mm DTT, and 0.1 mm CaCl2. The radioactivity of the eluate was determined by a liquid scintillation counter. Nonspecific radioactivity was determined in the presence of 50 μm nonradioactive ryanodine. By using this assay system, we were able to determine the ryanodine binding activity with only 20 ng of the purified RyR protein, which is one-tenth the minimum amount for the conventional filtration assay. [3H]Ryanodine binding to the purified RyR1 was determined under the identical condition to the filtration assay with polyethyleneimine-treated glass filters as described (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). The structure of RyR molecules was examined by electron microscopy after negative staining. It was necessary to completely eliminate phospholipid and to reduce detergent from the preparation, since their presence hampered the visualization of structural details of the proteinaceous receptors. To meet such requirements, the materials for the structural study were treated as follows. The RyR3 immunoprecipitated by anti-RyR3 beads was washed with a buffer containing 0.1% CHAPS, instead of 1% CHAPS and 0.5% egg lecithin, and eluted by the RyR3-peptide. Then, the peptide was removed by passing the solution through a Centri-sep column pre-equilibrated with a buffer containing 0.5 m NaCl, 5 mmsodium phosphate (pH 7.2), 0.1 m sucrose, 0.1% CHAPS, and 2 mm DTT (buffer C). For RyR1, the initial buffer was exchanged with buffer C by gel filtration on a Superose 6 column (Amersham Pharmacia Biotech). Those procedures did not deteriorate the [3H]ryanodine binding activity of RyRs unless the preparations were left standing for a long period. For examination by electron microscopy, 2–3 μl of the sample was applied to fresh thin carbon film over a 200-mesh copper grid. After 3 min, the residual solution was thoroughly rinsed off with the above buffer without CHAPS, and the sample was stained negatively with 1% uranyl acetate containing bacitracin (23Katayama E. J. Biochem. (Tokyo). 1989; 106: 751-770Crossref PubMed Scopus (46) Google Scholar). Specimens were set in a side entry goniometer for the JEOL 2000EX electron microscope with the carbon side facing upward. Stereo-paired micrographs were taken by tilting ±10° at 80 kV acceleration voltage (23Katayama E. J. Biochem. (Tokyo). 1989; 106: 751-770Crossref PubMed Scopus (46) Google Scholar). Location of the D2 segment in the RyR1 architecture was determined similarly by negative staining but with use of a specific anti-D2-region polyclonal antibody as a probe. Since the dimension of the IgG molecule is quite small and thin (∼150 kDa) as compared with the gigantic RyR molecule (∼2,200 kDa) standing upright on the carbon-support, it was anticipated that the IgG probe bound to the receptor molecule might be hard to identify by itself, in the thick stain layer embedding the total receptor assembly. Thus, we used a two-step procedure (24Wagenknecht T. Grassucci R. Berkowitz J. Wiederrecht G.J. Xin H.B. Fleischer S. Biophys. J. 1996; 70: 1709-1715Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 25Wagenknecht T. Radermacher M. Grassucci R. Berkowitz J. Xin H.B. Fleischer S. J. Biol. Chem. 1997; 272: 32463-32471Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) to search for the epitope site of the antibody, tentatively with IgG conjugated with a probe large enough to be easily found, and later with the antibody by itself, to finally pinpoint the precise site. The antibody against the D2 region (amino acid sequence 1358–1413) of RyR1 was produced with rabbits using GST-fusion protein as an antigen. A cDNA fragment (residues 4073–4239) was obtained from a cassette pBS-RyR1cs4 (26Tong J. Oyamada H. Demaurex N. Grinstein S. McCarthy T.V. MacLennan D.H. J. Biol. Chem. 1997; 272: 26332-26339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar) and cloned into the pGEX-3X vector (Amersham Pharmacia Biotech). Expression and purification of the GST-fusion protein was carried out according to the manufacturer's instructions. The IgG was affinity-purified by the RyR1-bound PVDF membranes (10Murayama T. Ogawa Y. J. Biochem. (Tokyo). 1992; 112: 514-522Crossref PubMed Scopus (75) Google Scholar). This antibody stained a single band of RyR1 on Western blot analysis of SR vesicles which, in turn, were specifically immunoprecipitated by the same antibody (data not shown). In order to produce a probe with a large marker, the antibody was biotinylated and conjugated with oligomeric avidin, a linear rod-like assembly. Biotinylation of the antibody was carried out by incubation of the antiserum with sulfo-NHS-LC-biotin (Pierce). An oligomeric avidin was prepared by addition of a divalent biotin (generous gift from Dr. Kazuo Sutoh to E. K.) to neutravidin (Pierce) at a molar ratio of 2:1 (divalent biotin/neutravidin) (27Yamamoto K. Tokunaga M. Sutoh K. Wakabayashi T. Sekine T. J. Mol. Biol. 1985; 183: 287-290Crossref PubMed Scopus (18) Google Scholar) and was mixed with the biotinylated antibody before use. For negative staining, purified RyR1 receptor was first applied onto a carbon film and left standing for 3 min. The grid was briefly washed and the solution containing the antibody with or without avidin conjugate was added. After 3–5 min incubation period, it was exhaustively washed with the buffer and stained negatively with uranyl acetate, as described above. Single channel recordings were carried out as described previously (28Oba T. Am. J. Physiol. 1997; 273: C1588-C1595Crossref PubMed Google Scholar). Briefly, lipid bilayer consisting of a mixture of l-α-phosphatidylethanolamine,l-α-phosphatidyl-l-serine, andl-α-phosphatidylcholine (5:3:2 by weight) in decane (40 mg/ml) was formed across a hole (∼200 μm in diameter) in a polystyrene partition separating two chambers referred to ascis (volume 1 ml) and trans (volume 1.5 ml). Thetrans chamber was held at virtual ground potential, and thecis chamber was voltage-clamped at −40 mV relative to the ground, unless noted otherwise. Incorporation of the purified RyR channel was performed in asymmetrical KCl solutions containing 500:50 mm (cis/ trans) KCl, 20 mmHEPES/Tris, pH 7.4, and 0.1 mm CaCl2. The protein was added to the cis chamber. After confirming the channel incorporation by the occurrence of flickering currents, further incorporation of the protein was prevented by supplement of an aliquot of 3 m KCl dissolved in 20 mm HEPES/Tris to thetrans chamber. Single channel currents were recorded in symmetrical solutions containing 500 mm KCl, 20 mm HEPES/Tris, pH 7.4, and various concentrations of free Ca2+ buffered with 1 mm EGTA. Free Ca2+ was calculated using the apparent binding constant of EGTA for Ca2+ by Harafuji and Ogawa (22Harafuji H. Ogawa Y. J. Biochem. (Tokyo). 1980; 87: 1305-1312Crossref PubMed Scopus (346) Google Scholar) and was confirmed by potentiometry with a handmade ETH1001-based Ca2+electrode. Only bilayers containing a single channel were used in this study. Experiments were carried out at room temperature (18–22 °C). We found that the purified RyRs could be incorporated into the bilayers in either orientation. The sidedness of the single channel was determined by response to Ca2+, because the RyR channel sensitively responded to cytoplasmic Ca2+. About 90% of the RyR channels was blocked by lowering the cis free Ca2+ to 10–100 nm with EGTA, indicating that the cytoplasmic side of most channels faced the cischamber. Single channel currents amplified by an Axopatch 1D patch clamp amplifier (Axon Instrument, CA) were displayed on an oscilloscope, filtered at 1 kHz using an eight-pole low-pass Bessel filter, and digitized at 5 kHz for analysis. Data were saved on the hard disk of an IBM personal computer. P o and the lifetime of open and closed events from records of duration >2 min were calculated by 50% threshold analysis using pClamp (version 6.0.4) software. The results were presented as means ± S.E. RyR3 was purified from rabbit diaphragm using immunoprecipitation with an antibody specific to RyR3 (anti-RyR3) (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) which was raised against a synthetic peptide corresponding to residues 4375–4387 of rabbit RyR3 (RyR3-peptide) (see "Experimental Procedures"). The diaphragm was found to express about 10-fold more abundant RyR3 than brain in rabbits (13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). As shown in Fig. 1 A, SR protein prepared from rabbit diaphragm contained a large amount of RyR1 that was easily seen on Coomassie Brilliant Blue-stained gel and positively detectable on Western blot with anti-RyR1 monoclonal antibody XA7 and a low level of RyR3 that was identified on anti-RyR3 blot. After solubilization with CHAPS, SR proteins were incubated with anti-RyR3-agarose beads to precipitate RyR3 (12Murayama T. Ogawa Y. J. Biol. Chem. 1996; 271: 5079-5084Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 13Murayama T. Ogawa Y. J. Biol. Chem. 1997; 272: 24030-24037Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The band for RyR3 was clearly detected in the precipitate on both protein staining and anti-RyR3 blot (Fig. 1 B, left lanes). Addition of 30 μm RyR3-peptide during incubation failed to precipitate RyR3 (Fig. 1 B, right lanes), indicating specific interaction of RyR3 with the anti-RyR3. RyR1 band was detected neither on protein staining (Fig. 1 B, left panel) nor on anti-RyR1 blot (data not shown, but see Fig. 2 B). RyR3 was thus effectively concentrated and separated from the other SR proteins. The immunoprecipitated RyR3 was then dissociated from the antibody by incubation with excess RyR3-peptide. Preliminary attempts of treatment with 0.1 m glycine HCl (pH 2.8) or 5m potassium thiocyanate, which is conventionally used to release antigen from antibody, caused total loss of [3H]ryanodine binding activity, indicating irreversible degeneration of RyR3 (data not shown). As shown in Fig. 1 C, RyR3 (arrowhead) was detected in supernatant after incubation with 30 μm RyR3-peptide, wher
The human leukemia cell lines HL-60, KG-1, KLM-2, ML-3, THP-1, and U-937 were treated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA partially or completely inhibited the proliferative activity of the cell cultures. The number of cells with the ability to reduce nitroblue tetrazolium increased in the TPA-treated cell lines HL-60, ML-3, THP-1, and U-937, whereas the cell lines KG-1 and KLM-2 remained nitroblue tetrazolium negative. Except for KG-1 and KLM-2, all TPA-treated cell lines showed varying degrees of strong adherence to plastic surface. The carboxylic esterase, acid phosphatase, hexosaminidase, and lactate dehydrogenase isoenzyme profiles from these cell lines were analyzed by isoelectric focusing on horizontal polyacrylamide gels. The new or stronger expression of an esterase isoenzyme which is specific for monocytes-macrophages was induced in HL-60, ML-3, THP-1, and U-937 but not in KG-1 or KLM-2. The new expression of the tartrate-resistant acid phosphatase isoenzyme was induced in ML-3, THP-1, and U-937. The number of esterase and acid phosphatase isoenzymes and the staining intensity of isoenzymes characteristic for myeloid cells were increased by TPA in all cell lines. The loss of the hexosaminidase I isoenzyme which is a marker of immature hematopoietic cells was noted in KG-1, ML-3, THP-1, and U-937. TPA triggered an increase in number and staining intensity of the lactate dehydrogenase isoenzymes in all cell lines. Some isoenzymatic changes (e.g., monocyte-specific esterase, tartrate-resistant acid phosphatase, hexosaminidase I) appear to correlate with TPA-induced differentiation while other alterations in the isoenzyme patterns do not (e.g., lactate dehydrogenase, other esterase and acid phosphatase isoenzymes). Differentiation of nonmonocytoid cells appears, at the isoenzyme level, to be quite different from that of the monocytoid cell lines.
Anti-ryanodine receptor (RyR) antibodies were measured in sera from 33 myasthenia gravis (MG) patients using three peptides from the human RyR1 sequence, two C-terminal peptides included in the functional calcium release channel, and an N-terminal peptide implicated in ion-conduction. Antibodies were more frequently positive against the two C-terminal peptides, particularly in thymoma-associated MG. In a preliminary open trial with FK506, immunosuppressant and enhancer of RyR-related sarcoplasmic calcium release, the authors observed the sustained benefits in anti-RyR-positive MG patients.
Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. Natural cPA and its chemically stabilized cPA derivative, 2-carba-cPA (2ccPA), inhibit chronic and acute inflammation, and 2ccPA attenuates neuropathic pain. Osteoarthritis (OA) is a degenerative disease frequently associated with symptoms such as inflammation and joint pain. Because 2ccPA has obvious antinociceptive activity, we hypothesized that 2ccPA might relieve the pain caused by OA. We aimed to characterize the effects of 2ccPA on the pathogenesis of OA induced by total meniscectomy in the rabbit knee joint.Intra-articular injection of 2ccPA (twice a week for 42 days) significantly reduced pain and articular swelling. Histopathology showed that 2ccPA suppressed cartilage degeneration in OA. We also examined the effects of 2ccPA on the inflammatory and catabolic responses of human OA synoviocytes and chondrosarcoma SW1353 cells in vitro. 2ccPA stimulated synthesis of hyaluronic acid and suppressed production of the metalloproteinases MMP-1, -3, and -13. However, it had no effect on the production of interleukin (IL)-6, an inflammatory cytokine. The suppressive effect of 2ccPA on MMP-1 and -3 production in synoviocytes and on MMP-13 production in SW1353 cells was not mediated by the lysophosphatidic acid receptor, LPA1 receptor (LPA1R).Our results suggest that 2ccPA significantly reduces the pain response to OA by inducing hyaluronic acid production and suppressing MMP-1, -3, and -13 production in synoviocytes and chondrocytes.
