Figure 3.Chromatographic elution properties of 3-nitrotyrosine modified muscle creatine kinase are altered.
(A)
Immunoblots of Blue Sepharose fractions of CKm show that fractions 9-12 are
tyrosine nitrated. CKm (1 μg, > 85 % pure) was resolved
on a denaturing SDS polyacrylamide gel, transferred to a PVDF membrane, and
analyzed by immunoblot using anti-3NT-antibody. (B) Immunoblots of
Blue Sepharose fractions 3-7 shows that 3-NT CKm is not detected in these
fractions. Blots probed with an anti-3-nitrotyrosine antibody (top panel)
detected nitrated glycogen phosphorylase in middle aged and aged muscle.
However, the purified CKm in this fraction is not nitrated. Blots were
reprobed with an anti-CK antibody (3B, lower panel). (C) Fractions
9-11 were further fractionated by reverse phase chromatography. These
fractions were spotted on to PVDF membranes and analyzed for nitrated CKm
using anti-nitrotyrosine antibody, and carbonylated CKm using anti-DNPH
antibody (D).
