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Volumn 273, Issue 5278, 1996, Pages 1078-1085

A crosslinked cofactor in lysyl oxidase: Redox function for amino acid side chains

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN LYSINE 6 OXIDASE; QUINONE DERIVATIVE;

EID: 0029821437     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5278.1078     Document Type: Article
Times cited : (315)

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    • max) of 454 nm. Typically, the stoichiometry of labeling is about 40 to 50 percent after correction for the presence of the second band in the LO preparation.
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    • note
    • 3 (pH 8.0) containing 2 M urea at 3°C, with shaking. Proteolytic digestion was initiated by the addition of thermolysin to 4 percent (w/w). A second portion of the protease was added after 24 hours. The digestion was stopped after 49 hours by cooling the solution at - 70°C.
  • 40
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    • note
    • 2O) with a linear gradient of 20 to 30 percent solvent B over 65 minutes at a flow rate of 1 ml per minute. Elution of peptides was monitored at 214 and 438 nm. The yield was 22 percent for the 36-minute peak based on radioactivity relative to that contained in the digestion solution.
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    • note
    • The predominant peak at 438 nm was lyophilized and redissolved in 50 mM sodium phosphate (pH 8.0). Subdigestion was initiated by the addition of Asp-N endoproteinase to a final concentration of 2.5 percent (w/w). The digestion mixture was incubated at 37°C with gentle shaking for 19 hours and later stored at - 7O°C. Peptides resulting from the digest were again separated on HPLC by a modification of a previously described procedure (5). A Vydac reversed-phase C18 column was used, and peptide elution was monitored at 220 and 438 nm. The single peak containing the active site peptide was collected, lyophilized, and subsequently used for sequencing, mass spectrometry, and resonance Raman studies. Overall yield for this single peak was about 26 percent relative to total peptides in the Asp-N digestion reaction. This corresponded to about 1.2 nmol of the active site peptide.
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    • note
    • Electrospray mass spectrum of the phenylhydrazine-derivatized LO active site peptide (sample 1) referred to as "basic structure" was acquired in an LC-ESIMS experiment with a Micromass BioQ quadrupole mass spectrometer equipped with an electrospray source. The mass spectrometer was scanned in noncontinuum mode over a range of m/z as 350 to 2000 at 5 s per scan.
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    • note
    • The accurate mass measurement was performed by Micromass AutoSpec SE spectrometry with etectrospray ionization on the triply charged ion of a cofactorcontaining peptide (sample 2) with doubly charged ions of bradykinin (m/z of 530.7885) and ceramicidin (m/z of 571.3608) as calibration standards.
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    • note
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    • Supported by NIH research grants GM39296 (J.P.K.), and GM27659 (D.M.D.), National Center for Research Resources Biomedical Research Technology Program grants P41 RR01614 (A.L.B.), and R37 AR 18880 and HL13262 (H.M.K.), and supported by the NSF Biological Instrumentation Program grant DIR 8700766 (A.L.B.).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.