Identification of a spectrally stable proteolytic fragment of human neutrophil flavocytochrome b composed of the NH2-terminal regions of gp91(phox) and p22(phox).
A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b 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 degrees 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 NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.
Foubert
T R
TR
Department of Microbiology, Montana State University, Bozeman, Montana 59717-3520, USA.
Bleazard
J B
JB
Burritt
J B
JB
Gripentrog
J M
JM
Baniulis
D
D
Taylor
R M
RM
Jesaitis
A J
AJ
D000596:Amino Acids / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006418:Heme / Q000032:analysis / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000737:chemistry*; D026901:Membrane Transport Proteins; D009252:NADPH Dehydrogenase / Q000737:chemistry*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D017830:Octoxynol / Q000494:pharmacology; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016622:Silver Staining; D013997:Time Factors
D000596:Amino Acids; D003573:Cytochrome b Group; D003902:Detergents; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D006418:Heme; D017830:Octoxynol; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase