Degradation of neurofilament proteins by purified human brain cathepsin D.
Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one- or two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two- to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP break-down products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskeletal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.
D001921:Brain / Q000378:metabolism*; D002402:Cathepsin D; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D002540:Cerebral Cortex / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007106:Immune Sera; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D008970:Molecular Weight; D016900:Neurofilament Proteins; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism
D007106:Immune Sera; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D014404:Tubulin; D002403:Cathepsins; D002402:Cathepsin D