FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2079832022> ?p ?o ?g. }

• W2079832022 endingPage "38861" @default.
• W2079832022 startingPage "38852" @default.
• W2079832022 abstract "A heme-bearing polypeptide core of human neutrophil flavocytochrome b558 was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochromeb that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60–66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 °C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60–66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH2-terminal 320–363 amino acid residues of gp91phox and the NH2-terminal 169–171 amino acid residues of p22phox. These findings provide direct evidence that the primarily hydrophobic NH2-terminal regions of flavocytochrome b are responsible for heme ligation. A heme-bearing polypeptide core of human neutrophil flavocytochrome b558 was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochromeb that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60–66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 °C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60–66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH2-terminal 320–363 amino acid residues of gp91phox and the NH2-terminal 169–171 amino acid residues of p22phox. These findings provide direct evidence that the primarily hydrophobic NH2-terminal regions of flavocytochrome b are responsible for heme ligation. phagocyte oxidase polyacrylamide gel electrophoresis chronic granulomatous disease diode array detector dithiothreitol glutamic acid, carboxyl-terminal side high performance liquid chromatography monoclonal antibody absorbance maximum monoclonal antibody octyl glucoside, octyl β-glucopyranoside 3,3′,5,5′-tetramethylbenzidine staphylococcal V8, endoproteinase Glu-C phenylmethylsulfonyl fluoride The NADPH oxidase of human neutrophils is a multisubunit, membrane-associated complex that is crucial for host protection against invading pathogens (1Nauseef W.M. Volpp B.D. McCormick S. Leidal K.G. Clark R.A. J. Biol. Chem. 1991; 266: 5911-5917Abstract Full Text PDF PubMed Google Scholar, 2DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (457) Google Scholar, 3Wientjes F.B. Segal A.W. Semin. Cell Biol. 1995; 6: 357-365Crossref PubMed Scopus (94) Google Scholar, 4Leusen J.H.W. Verhoeven A.J. Roos D. J. Lab. Clin. Med. 1996; 128: 461-476Abstract Full Text PDF PubMed Scopus (102) Google Scholar, 5Rotrosen D. Yeung C.L. Katkins J.P. J. Biol. Chem. 1993; 268: 14256-14260Abstract Full Text PDF PubMed Google Scholar, 6Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 7Nauseef W.M. Proc. Assoc. Am. Physicians. 1999; 111: 373-382Crossref PubMed Scopus (85) Google Scholar). The redox center and the only membrane-spanning component of the oxidase is flavocytochromeb558 (also known as flavocytochrome b, cytochrome b558, cytochrome b559), a heterodimeric protein composed of an extensively glycosylated, 91-kDa large subunit (570 amino acid residues), gp91phox,1and a 22-kDa non-glycosylated small subunit (192 amino acid residues), p22phox (8Segal A.W. Nature. 1987; 326: 88-91Crossref PubMed Scopus (209) Google Scholar, 9Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (314) Google Scholar). Intracellular binding sites for both FAD and NADPH have been identified on gp91phox (10Segal A.W. West I. Wientjes F.B. Nugent J.H.A. Chavan A.J. Haley B. Garcia R.C. Rosen H. Scrace G. Biochem. J. 1992; 284: 781-788Crossref PubMed Scopus (291) Google Scholar, 11Yoshida L.S. Chiba T. Kakinuma K. Biochim. Biophys. Acta. 1992; 1135: 245-252Crossref PubMed Scopus (10) Google Scholar, 12Rotrosen D. Yeung C.L. Leto T.L. Malech H.L. Kwong C.H. Science. 1992; 256: 1459-1462Crossref PubMed Scopus (315) Google Scholar, 13Doussière J. Brandolin G. Derrien V. Vignais P.V. Biochemistry. 1993; 32: 8880-8887Crossref PubMed Scopus (49) Google Scholar, 14Doussière J. Buzenet G. Vignais P.V. Biochemistry. 1995; 34: 1760-1770Crossref PubMed Scopus (44) Google Scholar). Flavocytochrome b functions as the terminal electron carrier prior to reduction of extracellular molecular oxygen to the antimicrobial precursor, superoxide anion (O⨪2) (2DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (457) Google Scholar,15Isogai Y. Iizuka T. Shiro Y. J. Biol. Chem. 1995; 270: 7853-7857Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 16Segal A.W. J. Clin. Invest. 1996; 83: 1785-1793Crossref Scopus (205) Google Scholar, 17Schrezel J. Serrander L. Banfi B. Nüβe O. Fouyouzi R. Lew D.P. Demaurex N. Krause K.H. Nature. 1998; 392: 734-737Crossref PubMed Scopus (166) Google Scholar, 18Jesaitis A.J. J. Immunol. 1995; 155: 3286-3288PubMed Google Scholar). Flavocytochrome b has also been proposed to function as a voltage-gated proton transporter that maintains intracellular pH and membrane potential during the oxidative burst (19Henderson L.M. Meech R.W. J. Gen. Physiol. 1999; 114: 771-786Crossref PubMed Scopus (56) Google Scholar,20Henderson L.M. Chappell J.B. Jones O.T. Biochem. J. 1987; 246: 325-329Crossref PubMed Scopus (276) Google Scholar). The characteristic absorbance spectrum of flavocytochrome bis attributed to the presence of heme prosthetic groups that are non-covalently coordinated by histidine residues within the protein. Due to the tenuous nature of this ligation scheme, the heme spectrum is lost under conditions that separate the individual subunits (21Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. Biochim. Biophys. Acta. 1988; 932: 71-83Crossref PubMed Scopus (53) Google Scholar), although heme remains associated with both subunits during electrophoresis at low temperature with lithium dodecyl sulfate-PAGE (22Quinn M.T. Mullen M.L. Jesaitis A.J. Clin. Res. 1991; 39: 353Google Scholar). Additionally, the detergent-solubilized protein is highly susceptible to proteolysis by the numerous endogenous phagocyte proteinases. These and other factors have prevented direct identification of the regions of the flavocytochrome bheterodimer that are responsible for heme coordination. There are, however, several lines of indirect evidence deriving from mutational (23Tsuda M. Kaneda M. Sakiyama T. Inana I. Owada M. Kiryu C. Shiraishi T. Kakinuma K. Hum. Genet. 1998; 103: 377-381Crossref PubMed Scopus (16) Google Scholar, 24Bolscher B.G. de Boer M. de Klein A. Weening R.S. Roos D. Blood. 1991; 77: 2482-2487Crossref PubMed Google Scholar) and spectroscopic analyses (25Fujii H. Yonetani T. Miki T. Kakinuma K. J. Biol. Chem. 1995; 270: 3193-3196Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 26Fujii H. Finnegan M.G. Miki T. Crouse B.R. Kakinuma K. Johnson M.K. FEBS Lett. 1995; 377: 345-348Crossref PubMed Scopus (18) Google Scholar) that implicate regions of gp91phox with heme binding. Inferences have also been made of a stacked heme orientation within the membrane bilayer, coordinated by two transmembrane helices of gp91phox based on similarities in primary sequence, spectral properties, and redox potentials between flavocytochrome band select members of the ferredoxin-NADP+ reductase family, including FRE1 ferric reductase of Saccharomyces cerevisiae (27Finegold A.A. Shatwell K.P. Segal A.W. Klausner R.D. Dancis A. J. Biol. Chem. 1996; 271: 31021-31024Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 28Taylor W.R. Jones D.T. Segal A.W. Protein Sci. 1993; 2: 1675-1685Crossref PubMed Scopus (108) Google Scholar, 29Shatwell K.P. Dancis A. Cross A.R. Klausner R.D. Segal A.W. J. Biol. Chem. 1996; 271: 14240-14244Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 30Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Crossref PubMed Scopus (282) Google Scholar). Likewise, heme localization within gp91phox has been inferred based on similarities to heme-coordinating regions of cytochrome P450 of Pseudomonas putida (31Poulos T.L. Finzel B.C. Gunsalus I.C. Wagner G.C. Kraut J. J. Biol. Chem. 1985; 260: 16122-16130Abstract Full Text PDF PubMed Google Scholar) and the β-subunit of cytochromeb559 of Synechocystis 6803 photosystem II (32Pakrasi H.B. De Ciechi P. Whitmarsh J. EMBO J. 1991; 10: 1619-1627Crossref PubMed Scopus (76) Google Scholar). These arguments and the intrinsic hydrophobic nature of the heme molecule would suggest placement within the NH2-terminal putative membrane-spanning regions of flavocytochrome b 2D. Baniulis, J. Burritt, and A. Jesaitis, unpublished observations. (33Royer-Pokora B. Kunkel L.M. Monaco A.P. Goff S.C. Newburger P.E. Baehner R.L. Cole F.S. Curnutte J.T. Orkin S.H. Nature. 1986; 322: 32-37Crossref PubMed Scopus (594) Google Scholar, 34Dinauer M.C. Orkin S.H. Brown R. Jesaitis A.J. Parkos C.A. Nature. 1987; 327: 717-721Crossref PubMed Scopus (266) Google Scholar, 35Teahan C. Rowe P. Parker P. Totty N. Segal A.W. Nature. 1987; 327: 720-721Crossref PubMed Scopus (192) Google Scholar), although there is no direct evidence to support this assumption. The intent of this study was to isolate and identify proteinase-stable heme-ligating regions of flavocytochrome b. Partially purified flavocytochrome b was exposed to staphylococcal V8 proteinase (endoproteinase Glu-C). HPLC size exclusion chromatography was then used to isolate proteolytic fragments that retained the characteristic 414-nm heme absorbance spectrum. With this approach, we successfully isolated a spectrally native polypeptide core that was then characterized by amino acid sequencing and immunoblotting. The fragment was found to be a heterodimer composed of the NH2-terminal 336–363 amino acid residues of gp91phox and the NH2-terminal 169–171 amino acid residues of p22phox. The core fragment retained 74% of the native heme absorbance, suggesting that it contained all of the heme-ligating regions of flavocytochrome b. These findings suggest that the hemes are positioned intra- or juxtamembrane within the NH2-terminal predicted transmembrane-spanning regions of the protein. KCl, NaCl, EDTA, NaN3, silver nitrate, sodium carbonate, gelatin, Trizma (Tris base), EGTA, and syringe filters (Whatman, 25-mm diameter, polyethersulfone membrane, 0.2-µm pore size) were purchased from Fisher. GlcNAc,N,N′-diacetylglucosamine (chitobiose), heparin-Sepharose® 4B beads, N-formyl-Met-Leu-Phe, dihydrocytochalasin B, Na2ATP, chymostatin, wheat germ agglutinin, diisopropyl fluorophosphate, sodium dithionite, bovine serum albumin, Hanks' balanced salts, MgCl2, NaH2PO4, Trizma-HCl (Tris-HCl), hemin chloride (bovine), hydrogen peroxide 30% (w/w) solution, lithium dodecyl sulfate, pyridine (HPLC grade), glutathione, free acid, reduced form, thioglycolic acid (thioglycollate, mercaptoacetic acid), free acid, trifluoromethanesulfonic acid, iodoacetamide, 3,3′,5,5′-tetramethylbenzidine (TMBZ) (free base), and proteinase inhibitor mixture (P8340) were from Sigma. Polyvinylidene difluoride membrane (0.2-µm pore size) was from Bio-Rad. Triton X-100 detergent was from EM Sciences Co. or Sigma. HEPES, Gammabind®, and CNBr-activated Sepharose 4B beads were purchased from Amersham Pharmacia Biotech. Ultrapure SDS was purchased from U. S. Biochemical Corp. N-Octyl-β-d-glucopyranoside (OG, octyl glucoside), dithiothreitol (DTT, Cleland's reagent), and phenylmethylsulfonyl fluoride (PMSF), were from Calbiochem-Novabiochem. The BCA protein assay and BlueRanger® prestained molecular weight markers for SDS-PAGE were from Pierce. Other prestained molecular weight markers were supplied by Life Technologies, Inc. Endoproteinase Glu-C (proteinase V8 salt-free, lyophilized, sequencing grade) from Staphylococcus aureus V8 was from Roche Molecular Biochemicals. Nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase developer kit for immunoblots was from Kirkegaard & Perry Laboratories (Gaithersburg, MD). The following buffers were used in this work: heparin wash buffer: 50 mm NaH2PO4, 1 mm EGTA, 1 mm MgCl2, 0.1% (v/v) Triton X-100, 2 mm NaN3, pH 7.4, supplemented with 0.1 mm DTT, 10 µg/ml chymostatin, 0.2 mmPMSF (final concentrations) just prior to use; heparin elution buffer: either 75 mm or 2.0 m NaCl, 50 mmNaH2PO4, 2 mm NaN3, pH 7.4, 1 mm EGTA, 1 mm MgCl2, 0.1% (v/v) Triton X-100, supplemented with 0.1 mm DTT, 10 µg/ml chymostatin, 0.2 mm PMSF (final concentrations) immediately prior to use; HPLC column buffer: 150 mm NaCl, 50 mm NaH2PO4, 1 mmEGTA, 1 mm MgCl2, 0.1% (v/v) Triton X-100, 2 mm NaN3, 0.1 mm DTT, pH 7.4; TS buffer; 200 mm Tris base, 2% SDS, pH 8.0. Isolation of human polymorphonuclear leukocytes from whole blood and purification of flavocytochrome b was carried out as described previously (9Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (314) Google Scholar, 36Quinn M.T. Parkos C.A. Jesaitis A.J. Methods Enzymol. 1995; 255: 477-487Google Scholar) or with the following modifications. The wheat germ agglutinin affinity steps were eliminated to reduce the degree of sample handling, thus resulting in higher recoveries of flavocytochrome b without having an apparent effect on the overall purity. After loading, the heparin column containing the flavocytochrome b was washed with 10–20 bed volumes of heparin wash buffer and eluted using either a 50 mm to 2.0m NaCl gradient or a 6-ml bolus of 1.0 m NaCl, both in heparin elution buffer. 1-ml fractions were collected, and the peak flavocytochrome b-containing fractions were pooled and concentrated to a final volume of ∼1 ml using a 30-kDa nominal molecular weight cutoff centrifugal concentration device. As a final purification step prior to digestion, the retentate was subjected to an HPLC size exclusion chromatography step, as described below, and collected in 400-µl aliquots. Aliquots that were reserved for predigested controls had PMSF and chymostatin added to 1 mmand 10 µg/ml, respectively. Beginning with the solubilization step, all purification and digestion steps were accomplished in 1 day with all samples kept on wet ice prior to digestion. Flavocytochromeb heme content prior to heparin purification was quantitated using the reduced minus oxidized spectrum at 558 nm using Δε558 = 29.3 (mm cm)−1 (37Lutter R. Van Shaik M.L.J. Van Zwieten R. Wever R. Roos D. Hamers M.N. J. Biol. Chem. 1985; 260: 2237-2244Abstract Full Text PDF PubMed Google Scholar), blanked against control buffer, and reduced by addition of freshly mixed sodium dithionite in deionized water to a final concentration of 10 mm. After heparin purification, flavocytochromeb heme quantitation was carried out using ε414= 130.8 (mm cm)−1 (37Lutter R. Van Shaik M.L.J. Van Zwieten R. Wever R. Roos D. Hamers M.N. J. Biol. Chem. 1985; 260: 2237-2244Abstract Full Text PDF PubMed Google Scholar) blanked against buffer. Absorbance values were determined using either a Hewlett-Packard HP 8452A diode array UV-visible spectrophotometer or a Molecular Dynamics Spectra-Max 250, environmentally controlled, 96-well microtiter plate, UV-visible spectrophotometer. When necessary, the samples were sonicated using either a Fisher 50 probe style Sonic Dismembrator, model XL2005, or a Fisher brand bath sonicator, model FS30. All HPLC analyses were carried out using a Hitachi, LS-6200 HPLC with an F-1050 fluorescence detector connected in series with an L-7450A UV-visible Diode Array Detector. Size exclusion chromatography was performed using a Amersham Pharmacia Biotech Superdex 200 HR®, 10–30 column, maintained at 4 °C with a flow rate of 0.4 ml/min, equilibrated for a minimum of 1.5 h prior to sample injection. The column was calibrated using size exclusion chromatography standards (Bio-Rad catalog number 1511901) that included thyroglobulin, 670 kDa; γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa, and vitamin B12, 1.35 kDa. The standard curve was fitted using a non-linear regression algorithm (GraphPad Prism version 3.01 for Windows 95, GraphPad Software, San Diego, www.graphpad.com). Prior to HPLC size exclusion chromatography, all samples were bath sonicated for 1–2 s and passed through a 0.2-µm pore size syringe filter. HPLC elution fractions were collected from the column in 400-µl aliquots at 1-min intervals. Time point samplings for V8 digestions were removed from the digestion vessel and placed on wet ice with proteinase inhibitors prior to separation by HPLC size exclusion using the same procedures as for intact flavocytochrome b. All absorbance values were corrected for dilution and differences in HPLC injection volumes. Lyophilized V8 proteinase was reconstituted at 50–500 µg/ml in either distilled water or HPLC column buffer and added to the partially purified flavocytochromeb samples, while mixing, to a final ratio of 1:15 (w/w), proteinase to flavocytochrome b heme (assuming a flavocytochrome b heterodimer molecular mass of 110 kDa). The digestion was carried out at 37 °C in a continuously stirred vessel and terminated by placement on wet ice and addition of PMSF and chymostatin to final concentrations of 1 mm and 10 µg/ml, respectively. Digested samples were stored on ice at 4 °C until further analyses were conducted. Initial quantities of purified flavocytochrome b used for digestion ranged between 1.8 and 2.7 nmol of heme. The fractions retained from the HPLC runs had additional PMSF and chymostatin added to the same concentrations as above and were kept on ice until further analyses were conducted. Heme quantitation of HPLC fractions was carried out by modifying the procedure originally described (38Thomas P.E. Ryan D. Levin W. Anal. Biochem. 1976; 75: 168-176Crossref PubMed Scopus (896) Google Scholar, 39Holland V.R. Saunders B.C. Rose F.L. Walpole A.L. Tetrahedron. 1974; 30: 3299-3302Crossref Scopus (169) Google Scholar) to include addition of ethanol to the assay mixture to a nominal concentration of 50% (v/v). Assays were conducted in 96-well microtiter plates, and absorbance values were measured with a Molecular Dynamics Spectra-Max 250 UV-visible, environmentally controlled microtiter plate spectrophotometer. Reagents used in the assay were prepared immediately prior to use. The short incubation times combined with the large number of samples that were simultaneously tested required the use of a multitipped pipette to reduce timing errors in the mixing of the individual microtiter plate wells. Hemin standard solutions were prepared for initial quantitation as reduced, alkaline pyridine-solubilized hemochrome (37Lutter R. Van Shaik M.L.J. Van Zwieten R. Wever R. Roos D. Hamers M.N. J. Biol. Chem. 1985; 260: 2237-2244Abstract Full Text PDF PubMed Google Scholar) by addition of 0.5 n NaOH to a final sample concentration of 0.075n and 4.0 m pyridine to 2.1 m final concentration. The standards were then reduced by addition of sodium dithionite to a final concentration of 10 mm and quantitated using the reduced-oxidized molar extinction coefficient, Δε556–540 = 20.7 (mm cm)−1(37Lutter R. Van Shaik M.L.J. Van Zwieten R. Wever R. Roos D. Hamers M.N. J. Biol. Chem. 1985; 260: 2237-2244Abstract Full Text PDF PubMed Google Scholar). TMBZ solution consisted of 10 mg of dry TMBZ mixed with 500 µl of glacial acetic acid, filtered through a 0.2 µm pore size polyethersulfone membrane filter. 15 µl of the TMBZ solution was mixed, per well, with 30 µl of hemochrome sample, 100 µl of absolute ethanol and incubated at ambient temperature for 2 min. At the end of the 2 min, 15-µl aliquots of 3% H2O2were added per well and the mixtures incubated at ambient temperature for 5 min. The absorbance was then measured at 660 nm, and the hemin concentration of the samples was determined relative to the standards. All absorbance values were corrected for dilution and differences in HPLC injection volumes. Flavocytochromeb containing fractions were resolved by SDS-PAGE using 5–20% acrylamide gradient gels (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar), and electrophoretic transfer of protein to nitrocellulose for immunoblotting was performed as described (41Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44923) Google Scholar). Anti-gp91phox primary antibodies used were mouse mAbs NL10, CL5, NL7, 3J. Burritt and A. Jesaitis, unpublished observations. and 54.1 (42Burritt J.B. Busse S.C. Gizachew B. Siemsen D.W. Quinn M.T. Bond C.W. Dratz E.A. Jesaitis A.J. J. Biol. Chem. 1998; 273: 24847-24852Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,43Burritt J.B. Quinn M.T. Jutila M.A. Bond C.W. Jesaitis A.J. J. Biol. Chem. 1995; 270: 16974-16980Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and anti-peptide rabbit polyclonal antibodies KIS2 and KQS (44Jesaitis A.J. Buescher E.S. Harrison D. Quinn M.T. Parkos C.A. Livesey S. Linner J. J. Clin. Invest. 1990; 85: 821-835Crossref PubMed Scopus (104) Google Scholar). Anti-p22phox primary antibodies used were mouse mAbs NS1, NS2, NS5, CS9,3 and 44.1 (42Burritt J.B. Busse S.C. Gizachew B. Siemsen D.W. Quinn M.T. Bond C.W. Dratz E.A. Jesaitis A.J. J. Biol. Chem. 1998; 273: 24847-24852Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 43Burritt J.B. Quinn M.T. Jutila M.A. Bond C.W. Jesaitis A.J. J. Biol. Chem. 1995; 270: 16974-16980Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar), the rabbit polyclonal antibodies, anti-peptide EAR (45Quinn M.T. Parkos C.A. Walker L.E. Orkin S.H. Dinauer M.C. Jesaitis A.J. Nature. 1989; 342: 198-200Crossref PubMed Scopus (181) Google Scholar), and R3179 (9Parkos C.A. Allen R.A. Cochrane C.G. Jesaitis A.J. J. Clin. Invest. 1987; 80: 732-742Crossref PubMed Scopus (314) Google Scholar) produced by injecting rabbits with intact flavocytochrome b. Nitrocellulose transfers probed with primary antibodies were then probed with goat anti-rabbit, or goat anti-mouse, alkaline phosphatase-conjugated secondary antibodies and visualized using the Kirkegaard & Perry Laboratories nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate developer kit. Silver staining of polyacrylamide gels was carried out by overnight rocking in 50% methanol, 12% acetic acid in water, followed by three 20-min washes in 50% ethanol/water. Gels were then drained, completely submerged for 1 min in a solution containing 0.2 g/liter sodium thiosulfate, and washed 3 times for 30 s each with distilled water. After draining, 100 ml of a solution containing 0.2 g/liter silver nitrate, 1.0 ml/liter formaldehyde in water was added, and the gels were rocked for 20–30 min. The gels were then rinsed three times with water for 30 s each and developed by addition of 200 ml of 60 g/liter sodium carbonate, 1.0 ml/liter 37% (w/w) formaldehyde, and 30 ml/liter of the 0.2 g/liter sodium thiosulfate in distilled water and stopped by addition of 25% isopropyl alcohol and 10% acetic acid in water. NH2-terminal sequence analyses by Edman degradation were performed by Harvard Microchem (Cambridge, MA), and the samples were prepared as per their recommendations. To reduce the quantity of ubiquitous keratin in the SDS-PAGE reagents, twice recrystallized SDS (46(1999) Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) p. 10.8.2, John Wiley & Sons Inc., New York.Google Scholar) and distilled water that had been filtered through a 10-kDa nominal molecular weight cutoff filter were used. All other aqueous reagents were filtered through a 0.2-µm pore size membrane filter. Inadvertent chemical modification of the NH2 termini of peptides to be sequenced was avoided by including the following modifications to the SDS-PAGE protocol (40Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar). 5–20% gradient polyacrylamide resolving gels were allowed to polymerize overnight at ambient temperature. The stacking gel was poured, allowed to polymerize, and then pre-run without sample for 45 min at 4 mA constant current in the presence of Running buffer containing 5 µmreduced glutathione. The glutathione-containing buffer was removed; the samples were loaded, and the electrophoresis was conducted at 40 mA constant current using new Running buffer that contained 100 µm thioglycolic acid (47Hunkapiller M.W. Lujan E. Ostrander F. Hood L.E. Methods Enzymol. 1983; 91: 227-236Crossref PubMed Scopus (684) Google Scholar). Following SDS-PAGE, the proteins were transferred to polyvinylidene difluoride membrane, stained with Amido Black for visualization (46(1999) Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) p. 10.8.2, John Wiley & Sons Inc., New York.Google Scholar), excised from the membrane, and the strips washed 3× by gentle vortexing for 15 s each in filtered distilled water. The individual strips were then air-dried and placed in sealed plastic tubes for shipment. The diffuse band centered at ∼90 kDa, and the consolidated bands at 22 kDa were excised for NH2-terminal sequence analyses of the nondigested flavocytochrome b. The single band at 17 kDa and the entire broad band from ∼50 to 70 kDa were excised from the polyvinylidene difluoride membrane for NH2-terminal sequence analyses of the 1-h digest fragments. Samples were reduced by addition of an equal volume of DTT solution (50 mm DTT in TS buffer) and heated 4–5 min at 90 °C. Alkylation was performed by adding 0.1 volume (sample + DTT mixture) of iodoacetamide stock (46 mg of iodoacetamide dissolved in 200 mm Tris base, pH 8.0), followed by incubation at 90 °C for 3–4 min with IgG used as a procedural control. Iodoacetamide was added without DTT treatment to sample controls. All samples were then added to sample loading buffer, separated by SDS-PAGE, and immunoblotted as described above. For chemical deglycosylation, both intact and digested polypeptides were precipitated by addition of 80% (v/v) trichloroacetic acid in distilled water to a final sample concentration of 15% (v/v). Samples were then cooled at −20 °C for 30 min, followed by centrifugation at 180,000 × g for 15 min. The supernatant fraction was removed, and the pellet was then washed twice in −20 °C acetone to remove the trichloroacetic acid and either allowed to air dry or purged under a stream of dry argon at room temperature. The pellet was then resuspended into 100 µl of neat trifluoromethanesulfonic acid (48Sojar H.T. Bahl O.P. Methods Enzymol. 1987; 138: 341-350Crossref PubMed Scopus (86) Google Scholar, 49Sojar H.T. Bahl O.P. Arch. Biochem. Biophys. 1987; 259: 52-57Crossref PubMed Scopus (79) Google Scholar) by bath sonication in sealed tubes, purged with argon, and incubated on ice for 3 h in a ventilation hood. At the end of the incubation, the samples were cooled to less than −20 °C by immersion in a mixture of dry ice and ethanol. Neat pyridine, likewise cooled to less than −20 °C, was then slowly added to terminate the reaction. The volatile organic phase was removed under a stream of dry argon at room temperature, and the resulting gel was resuspended in distilled water and dialyzed against several changes of 10 mm phosphate-buffered saline at 4 °C. The protein was precipitated by addition of trichloroacetic acid to 15% (v/v), incubated at −20 °C for 15 min, and pelleted by centrifugation at 20,000 × g for 15 min at 4 °C. The resulting pellets were washed twice with −20 °C neat acetone and pelleted each time by centrifugation at 20,000 × g for 10 min at 4 °C. Samples were then reduced and alkylated as described above, mixed with loading buffer, and added directly to the lanes for separation by SDS-PAGE. Enzymatic deglycosylation of flavocytochromeb was carried out using peptideN-glycosidase F HPLC size exclusion chromatography fractions were denatured by addition of SDS to 0.5% in the presence of 10 mm DTT and 1 µl/ml proteinase inhibitor mixture and then heated to 100 °C for 10 min. Reagents supplied by the manufacturer were then added as per their instructions and incubated at 37 °C for 1 h with intermittent mixing. All predicted mass analyses based on primary sequence were done using either General Protein Mass Analysis for Windows, version 4.04, Lighthouse Data, or Statistical Analysis of Protein Sequences (SAPS) (50Brendel V. Bucher P. Nourbakhsh I.R. Blaisdell B.E. Karlin S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2002-2006Crossref PubMed Scopus (342) Google Scholar), available at www.isrec.isb-sib.ch/software/SAPS. Molecular mass determination by SDS-PAGE was extrapola" @default.
• W2079832022 created "2016-06-24" @default.
• W2079832022 creator A5031182133 @default.
• W2079832022 creator A5035171748 @default.
• W2079832022 creator A5039396452 @default.
• W2079832022 creator A5055061299 @default.
• W2079832022 creator A5061764760 @default.
• W2079832022 creator A5072881759 @default.
• W2079832022 creator A5079948516 @default.
• W2079832022 date "2001-10-01" @default.
• W2079832022 modified "2023-10-12" @default.
• W2079832022 title "Identification of a Spectrally Stable Proteolytic Fragment of Human Neutrophil Flavocytochrome b Composed of the NH2-terminal Regions of gp91 and p22" @default.
• W2079832022 cites W117605187 @default.
• W2079832022 cites W1498347766 @default.
• W2079832022 cites W1535247289 @default.
• W2079832022 cites W1547482025 @default.
• W2079832022 cites W1548052056 @default.
• W2079832022 cites W1556152083 @default.
• W2079832022 cites W1559156419 @default.
• W2079832022 cites W1566004432 @default.
• W2079832022 cites W1578030198 @default.
• W2079832022 cites W1586099293 @default.
• W2079832022 cites W1639029418 @default.
• W2079832022 cites W1661985738 @default.
• W2079832022 cites W1810573536 @default.
• W2079832022 cites W1837854019 @default.
• W2079832022 cites W1886576719 @default.
• W2079832022 cites W1940122864 @default.
• W2079832022 cites W1964603858 @default.
• W2079832022 cites W1965104216 @default.
• W2079832022 cites W1966699554 @default.
• W2079832022 cites W1969217001 @default.
• W2079832022 cites W1977014164 @default.
• W2079832022 cites W1978727971 @default.
• W2079832022 cites W1983635965 @default.
• W2079832022 cites W1986370112 @default.
• W2079832022 cites W1989764592 @default.
• W2079832022 cites W1997502223 @default.
• W2079832022 cites W2004145071 @default.
• W2079832022 cites W2007437829 @default.
• W2079832022 cites W2008399824 @default.
• W2079832022 cites W2008671156 @default.
• W2079832022 cites W2013005096 @default.
• W2079832022 cites W2013070909 @default.
• W2079832022 cites W2018549431 @default.
• W2079832022 cites W2022942610 @default.
• W2079832022 cites W2025054558 @default.
• W2079832022 cites W2030225423 @default.
• W2079832022 cites W2030242320 @default.
• W2079832022 cites W2034290678 @default.
• W2079832022 cites W2034782642 @default.
• W2079832022 cites W2035995596 @default.
• W2079832022 cites W2039490296 @default.
• W2079832022 cites W2040096357 @default.
• W2079832022 cites W2041237554 @default.
• W2079832022 cites W2049832673 @default.
• W2079832022 cites W2050742607 @default.
• W2079832022 cites W2051794463 @default.
• W2079832022 cites W2058179975 @default.
• W2079832022 cites W2060193350 @default.
• W2079832022 cites W2061592631 @default.
• W2079832022 cites W2066657229 @default.
• W2079832022 cites W2076055514 @default.
• W2079832022 cites W2076075128 @default.
• W2079832022 cites W2079436411 @default.
• W2079832022 cites W2085794106 @default.
• W2079832022 cites W2095835743 @default.
• W2079832022 cites W2100837269 @default.
• W2079832022 cites W2101108802 @default.
• W2079832022 cites W2119518322 @default.
• W2079832022 cites W2139868634 @default.
• W2079832022 cites W2143908574 @default.
• W2079832022 cites W2163785575 @default.
• W2079832022 cites W2220912830 @default.
• W2079832022 cites W2237782819 @default.
• W2079832022 cites W2240990209 @default.
• W2079832022 cites W2399591470 @default.
• W2079832022 cites W2411636689 @default.
• W2079832022 cites W3023000517 @default.
• W2079832022 doi "https://doi.org/10.1074/jbc.m104373200" @default.
• W2079832022 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11504718" @default.
• W2079832022 hasPublicationYear "2001" @default.
• W2079832022 type Work @default.
• W2079832022 sameAs 2079832022 @default.
• W2079832022 citedByCount "19" @default.
• W2079832022 countsByYear W20798320222013 @default.
• W2079832022 crossrefType "journal-article" @default.
• W2079832022 hasAuthorship W2079832022A5031182133 @default.
• W2079832022 hasAuthorship W2079832022A5035171748 @default.
• W2079832022 hasAuthorship W2079832022A5039396452 @default.
• W2079832022 hasAuthorship W2079832022A5055061299 @default.
• W2079832022 hasAuthorship W2079832022A5061764760 @default.
• W2079832022 hasAuthorship W2079832022A5072881759 @default.
• W2079832022 hasAuthorship W2079832022A5079948516 @default.
• W2079832022 hasBestOaLocation W20798320221 @default.
• W2079832022 hasConcept C116834253 @default.
• W2079832022 hasConcept C153911025 @default.
• W2079832022 hasConcept C185592680 @default.

• next