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Volumn 274, Issue 5287, 1996, Pages 618-620

Enhanced dissociation of HLA-DR-bound peptides in the presence of HLA- DM

Author keywords

[No Author keywords available]

Indexed keywords

HLA DR ANTIGEN; MAJOR HISTOCOMPATIBILITY ANTIGEN CLASS 2; SYNTHETIC PEPTIDE;

EID: 0029824101     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5287.618     Document Type: Article
Times cited : (262)

References (27)
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    • note
    • d (purified with an MKD6 monoclonal antibody affinity column by parallel procedures) had no activity in peptide release assays, excluding a role for detergent or other components. Peptides were synthesized by fluorenyl methoxycarbonyl chemistry with a Ranin Symphony multiple peptide synthesizer and labeled with biotin at the α-amino group before or after deprotection and cleavage by reaction with biotin-amido caproate N-hydroxysuccinimide as described (14). All binding and dissociation reactions were done in 0.1 M citrate-phosphate buffer (pH 5,0) with 0.2% NP-40 containing protease inhibitors (binding buffer). DR-biotin-peptide complexes were formed by incubation of 500 nM DR with 5 μM biotinpeptide complex for 18 hours at 37°C. For dissociation, preformed complexes were diluted 1:10 and incubated with 100 μM unlabeled MAT(17-31) peptide fragment (sequence, SGPLKAEIAQRLEDV) (15) or HSP(3-13) with or without DM. After neutralization, the complexes were captured on L243-coated microtiter wells at 4°C and biotin-peptide complexes were measured with streptavidin-europium as described (8).
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    • note
    • 50 value of 1.0.
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    • note
    • Dissociation rates were measured in the absence of DM for most of the peptides represented in Fig. 2A. Complexes were formed by incubation of 500 nM purified DR1 or DR3 with 2.5 μM biotin-peptide for 18 to 72 hours. Samples were diluted and incubated with 100 μM HA(306-318) (DR1) or HSP(3-13) (DR3) in binding buffer at pH 5 and 37°C. At various time points, samples were placed on ice and the pH was neutralized with 1 M tris-HCl (pH 7.5) to prevent further dissociation. DR-biotin-peptide complexes were quantified by the europium fluorescence immunoassay.
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    • note
    • 2, [DRp·DM]. Under conditions where peptide dissociation is irreversible, application of the steady approximation gives equation presented
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    • note
    • Abbreviations for amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly: H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
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    • note
    • Solid-phase HA(306-318) was labeled through the α-amino group with fluorescein isothiocyanate (FITC) before deprotection and cleavage. Other peptides, including MBP(90-102) (sequence, HFFKNIVTPRTPA) (15), were labeled with FITC after cleavage. Preformed DR1-peptide complexes were diluted to 1 μM total DR1 and incubated at 37°C in 0.1 M citratephosphate buffer (pH 5) and 0.2% NP-40 in the presence of 0.1 to 0.2 μM unlabeed HA(306-318) and the indicated concentration of affinity-purified DM. At various times, 10-μl samples were removed and DR1-FITC-peptide complexes were quantified with a Tosohaas TSK GFC 200 high-performance size-exclusion column (7.8 × 150 mm) and a Shimadzu RF-10A fluorometer with 490-nm excitation and detection at 520 nm. The column buffer was 50 mM phosphate, 150 mM NaCl, 1 mM dodecyl β-D-maltoside (pH 7.0) (16). DR eluted between 3.2 and 3.8 min with a flow rate of 1 ml/min.
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    • note
    • We thank K. Wilkinson and L. Stern for valuable discussions and advice and J. Moore and E. Spotts for excellent technical support. Supported by grants AI30554 and AI33614 from NIH. D.A.W. received support from the Centers for Disease Control and Prevention.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.