PubMed · 7124519
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J Wyman. 1982. Linkage graphs.. https://doi.org/10.1007/978-1-4615-9281-5_3
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The polymerization of alpha1-antitrypsin within the hepatic cell leads to alpha1-antitrypsin deficiency. Both the conformational changes and the kinetics of the polymerization process are poorly understood. Here we describe fluorescence experiments investigating the polymerization reaction using the fluorescent probe4, 4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate (bis-ANS) which bound to both native and polymerized alpha1-antitrypsin. Biphasic changes in bis-ANS fluorescence were observed during formation of alpha1-antitrypsin polymers. Initially a rapid increase in fluorescence signal was observed; it was followed by a gradual reduction in fluorescence signal. The first phase is a conformational change in which the A beta-sheet of alpha1-antitrypsin opens, whereas the second phase represents the insertion of the reactive center loop into the A beta-sheet of another molecule and therefore determines the rate of the polymerization process.
When the quartz crystal microbalance (QCM) is operated in contact with solution and used to detect inertia increases caused by macromolecules binding to its surface, resonance frequency shifts are reported in the literature to be greater than, less than, and the same as an identical macromolecular mass would cause as a dry layer. A previous report of wet and dry M13 DNA giving the same, linear frequency versus mass response is examined. The M13 data are shown to follow the reciprocal of the square root of mass, not the reported linear relationship. New experiments on RNA duplexes oscillated in solution are reported. A lossy polymer layer is placed between the QCM and RNA. When changes in density, viscosity, and included water are eliminated, the response remains linear for a constant adlayer thickness. The expectation that response per unit mass should decrease with distance from the QCM surface is demonstrated. Total decoupling of mass lying beyond the acoustic overlayer is also demonstrated. The present results are placed in context with recently published results from a study of progressively thicker protein layers bound to the QCM.
The mammalian MCM protein family, presently with six members, exists in the nuclei in two forms, chromatin-bound and unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM (hMCM) proteins by immunoprecipitation. In the present study, we applied this procedure to HeLa cells synchronized in each of the G1, S, and G2/M phases and could detect hMCM heterohexameric complexes in all three. In addition, depending on the cell cycle and the state of chromatin association, hMCM2 and 4 in the complexes were found to variously change their phosphorylation states. Concentrating attention on G2/M phase hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good substrates for cdc2/cyclin B in vitro. Furthermore, when cdc2 kinase was inactivated in temperature-sensitive mutant murine FT210 cells, the G2/M hyperphosphorylation of the murine MCM2 and MCM4 and release of the MCMs from chromatin in the G2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of mechanisms negatively regulating the MCM complex-chromatin association.