Comparison of ∼110-kDa complex formation between wild-type (WT) parkin and PD-linked parkin variants

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> COS1 cells were transiently transfected with 70 ng/cm of WT or R256C mutant parkin cDNA, 50 ng/cm of A82E or K161N parkin cDNA, 30 ng/cm of K211N cDNA and 120 ng/cm of R275W cDNA. (A) Transfected COS1 cells were extracted in 1% Triton X-100, followed by glycerol gradient centrifugation of the extracts. Fractions 2–6 of the gradient were analysed by BN-PAGE and parkin immunoblotting to visualize monomer (arrowhead) and ∼110-kDa complex (arrow). (B) In the experiments shown in (A), the amounts of parkin complex and parkin monomer were quantified. The graph represents the amount of parkin complex, expressed as a percentage of the sum of parkin complex and monomer. Asterisks denote significant difference ( < 0.05) from WT. (C and D) In parallel with each of the experiments shown in (A and B), 15 µg of Triton-soluble protein extract was analysed with SDS–PAGE and parkin immunoblotting to compare soluble parkin protein levels between WT and mutant variants. At the low signal intensity of the blots shown in (C), parkin appeared as a doublet due to the presence of an N-terminally truncated parkin species generated through an internal translation initiation site (). No endogenous parkin signal could be observed by SDS–PAGE in untransfected (Untransf.) COS1 cells (C), except after very prolonged film exposures (not shown). The graph in (D) shows SDS–PAGE and Western blot quantification of the Triton-soluble parkin levels. Within each experiment, the level of the parkin mutants was normalized to that of WT parkin. There were no significant differences in soluble parkin levels between WT, A82E, K161N, K211N or R256C ( = 0.96 by one-way )

BN-PAGE reveals a stable, non-covalent, ∼110-kDa parkin complex in brain

<p><b>Copyright information:</b></p><p>Taken from "Parkin occurs in a stable, non-covalent, ∼110-kDa complex in brain"</p><p></p><p>The European Journal of Neuroscience 2008;27(2):284-293.</p><p>Published online Jan 2008</p><p>PMCID:PMC2253705.</p><p>© The Authors (2007). Journal Compilation © Federation of European Neuroscience Societies and Blackwell Publishing Ltd</p> (A and B) Extracts of brain stem and diencephalon from wild-type and parkin knockout (KO) mice were separated into pellet (P) and supernatant (S) fractions, as described in Materials and methods and . P and S fractions were further fractionated by glycerol gradient centrifugation. Twelve P and 12 S fractions (numbered from the top of the glycerol gradient to the bottom) were analysed by BN-PAGE, followed by Western blotting with either the polyclonal anti-parkin antibody CS2132 (A) or the monoclonal anti-parkin antibody PRK109 (B) The CS2132 antibody detected a 450–550-kDa band in fraction S6, which was also found in parkin-null extract (A). By contrast, the PRK109 antibody (B) revealed a band in fraction S5 at a lower molecular weight (indicated by the arrow), which was absent in parkin knockout brain. The PRK109 antibody also showed some minor, non-specific immunoreactivity at higher molecular weights (indicated by the asterisk) in S5 from both wild-type and parkin knockout. (C) The graph shows how BN gels were calibrated based on the relative mobilities of native protein size markers to determine the molecular weight (MW) of the parkin complex. Markers, denoted by black circles, were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa) and bovine serum albumin (67 kDa). The dotted line indicates the estimated MW of the parkin complex. (D) In the left and middle panels, heating brain fraction S5 at 100 °C in 1.5% sodium dodecyl sulphate (SDS) for 10 min immediately prior to BN-PAGE disrupted the ∼110-kDa parkin complex and led to the appearance of monomeric parkin (indicated by the arrow). In the rightmost panel, approximately 50 ng of purified recombinant parkin was analysed by BN-PAGE and Western blot with PRK109 without heat treatment or addition of SDS, revealing a band (indicated by the arrow) with apparent molecular weight (∼50 kDa) consistent with that of monomeric parkin. (E) Omission of Triton X-100 from the extraction protocol did not change the native molecular mass of the parkin complex from brain or the amount of parkin extracted. Numbers to the left of (A), (B), (D) and (E) indicate MWs of the native protein size markers