Interprotein disulfide bonds formed during isolation process tighten the structure of the postsynaptic density.
Postsynaptic densities (PSDs) isolated by biochemical means consist of a complex mixture of proteins that tightly bond to each other. The purpose of this report is to study whether the numerous interprotein disulfides found in the isolated PSDs contribute to the tight structure of the PSDs and whether these interprotein disulfides exist in vivo. PSDs were isolated from pig cerebral cortex by conventional methods except that iodoacetic acid (IAA) was added to all solutions to curtail the formation of disulfides during the isolation process. The PSDs thus isolated were fragmented easily by treatment with chaotropic reagents or ionic detergents, whereas the PSDs isolated in IAA-free solutions were resistant to these treatments. Electron microscopy revealed that the PSDs isolated in IAA-containing solutions were more fragmented than those isolated in IAA-free solutions. Furthermore, the PSD sample isolated in IAA-free solutions contained very large disulfide-linked aggregates that were virtually absent from the PSDs isolated in IAA-containing solutions. Our results suggest that the exceptionally tight structure of the PSDs isolated by conventional methods is due largely to the new disulfides formed during the isolation process and that the PSD proteins under in vivo conditions are held together primarily by noncovalent interactions.
Lai
S L
SL
Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chiang
S F
SF
Chen
I T
IT
Chow
W Y
WY
Chang
Y C
YC
D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000648:ultrastructure*; D003902:Detergents; D004220:Disulfides / Q000737:chemistry*; D007202:Indicators and Reagents; D019807:Iodoacetic Acid; D008745:Methylation; D008854:Microscopy, Electron; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry; D013552:Swine; D013569:Synapses / Q000737:chemistry* / Q000648:ultrastructure*
D003902:Detergents; D004220:Disulfides; D007202:Indicators and Reagents; D009419:Nerve Tissue Proteins; D019807:Iodoacetic Acid