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Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "activated" Ub, linked via a thioester bond to a specific cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the(More)
In an optimized reconstituted system, the basic kinetic properties of the phosphate carrier from bovine heart mitochondria, e.g. the influence of membrane potential, pH, and proton gradient, were investigated for the two physiological modes of transport (Pi-/Pi- antiport and electroneutral, unidirectional phosphate transport). On the basis of these data,(More)
The phosphate carrier from bovine heart mitochondria was reconstituted into liposomes by the removal of detergent using hydrophobic ion-exchange columns. Reversible blocking of the carrier function during chromatographic steps was possible by the application of the inhibitor p-(chloromercuri)benzenesulfonate at low temperature. Thus, both forward and(More)
Upon modification of the reconstituted aspartate/glutamate carrier by various amino acid-reactive chemicals a functional lysine residue at the exofacial binding site was identified. The inactivation of transport function by the lysine-specific reagents pyridoxal phosphate (PLP, IC50 400 microM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS,(More)
Treatment of the reconstituted aspartate/glutamate carrier from mitochondria with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (Nbd-Cl) led to complete inactivation of carrier function. Inhibition could be attributed to chemical modification of one single cysteine in the active site. This residue was specifically protected in the presence of aspartate or(More)
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