Figure 6.The 130 kDa CKm species is 3-nitrotyrosine modified in an age-dependent fashion. (A) Blue
Sepharose protein fractions #26-#32 (see Figure 5) that contained anti-CKm
immunoreactive proteins with apparent masses of 88 (CKm 88) and 130 (CKm
130) kDa were pooled from young, middle-aged and aged mouse samples and individually
loaded onto a 1 ml mono-Q-Sepharose column (Biorad Laboratories). After
application, samples were washed with 10 ml of 25 mM Tris pH 8.0, developed
with a shallow 20 ml linear NaCl gradient (0-250 mM NaCl in 25 mM Tris pH
8.0) followed by a steep 10 ml NaCl gradient (250 mM - 1M NaCl in 25 mM
Tris pH 8.0). Flow rate equaled 1 ml/min throughout purification and
fractions were collected at a rate of one fraction per minute. The above
chromatograph was obtained by fractionation of the middle-aged protein
sample, fractionation of the young and aged samples yielded similar
chromatographs. Fractions 26-28 (indicated on the figure) contained CKm
130. These fractions were pooled and analyzed for 3-nitrotyrosine
modification. (B) Pooled protein (0.5 μg) from young
(Y), middle-aged (M), and aged (A) Q-Sepharose fractionations were resolved
by SDS PAGE and transferred to a PVDF membrane. Blots probed with an
anti-3NT antibody [top blot, panel (B)] reveal that CK 130 is 3-NT modified
in an age dependent manner. The membrane was then stained with Coomassie
Blue [bottom blot, panel (B)] to normalize protein loading. (C)
Densitometry was used to compare the relative abundance of the 3-NT
modified form of CK 130 between age groups.
