The sensitivily of lipoamide dehydrogenase (dihydro1ipoaniide:NAD ' oxidoreductase E3) from Azotubacter vinelandii to inhibition by NADH requires measurement of the activity in lhc initial phase of the reaction. Stopped-flow turnover experiments show that k,,,, is 830 s-' compared with 420 S -' found in standard steady-state experiments,Mutations at the xi-side of the flavin prosthetic group that cause severe inhibition by NADH were studied. Tyrl6 was replaced by phenylalanine and serine, which muses the loss o f two intersubunit H-bonds.[F16]E3 shows only 5.7% of wild-type activity in the standard assay procedurc, but m a l y m l by stopped-flow the activity is 70% of the wild-type enzyme. The NADH-C1,Ind (dichloroindophenol) activity was normal or slightly increased. The inhibition by NADH is competitive with respect t o NAD', Ki = SO pM. Spectral analysis show that electrons readily pass over from the disulfide to the FAD, indicating an increase in the redox potential of the flavin. It is concluded that subunit interaction plays an irnportant role in the protection of the enzyme against over-reduction by decreasing the redox potential of the flavin.The interaction of wild-type o r mutant enzymcs with the core component of the pyruvate (E2p) or oxoglutarate (E2o) dehydrogenase multienzyme complex relieves the inhibition to a large extent. In the mutant enzymes, the mechanism of inhibition changes from competitive to the mixed-type inhibition observed for the wild-type enzyme.The stabilizing effect of E2 on [Fl6]E3 was used as an assay to analyze the stoichiumetry of interaction of E3 with E2p as well as E2o. 1 mol E2p monomer was sufficient to saturate 1 in01 E3 dirrier with a K,, of about 1 nM. Similarly, 1 mol E2o saturated the E3 dirner with a Kd of 30 nM. From these experiments it is concluded that the E3-binding domain of E2 interacts with the subunit intcrface of E3 near the dyad axis, thus preventing sterically thc interaction with a second molecule of the binding domain. This mode of interaction, which causes asymmetry in the complex, explains [he stabilization against over-reduction by tightening the subunit interaction.Subgene cloning of the E2p component of the pyruvaic dehydrogenase complex is described in order to obtain a complcx between the lipoamide dehydrogenase componenl (E3) and the binding domain of E2p. A unique restriction site in the DNA encoding the flexible linker between the third lipoyl domain and the binding domain combined with timed digestion with exonuclease Ru131 wits used to create a set of deletion mutants in the N-terminal region of the binding-catalytic didomain, fused to six N-terminal amino acids from 8-galactosidase. The expressed proteins, selected for E2p activity, were analyzed for binding of E3 and Elp. The shortest fusion protein containing a functional binding domain wiis expressed and purified. IF141E3 was combined with this fusion protein in a stoichiometric ratio and the rcsultiiig complex was subjected to limited proteolysis to remove the catalytic domain. The r...
The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.
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