From the possible direct requirement for ATP in degradation of proteins in bacteria, it was not too unlikely to assume a similar direct mechanism in the degradation of cellular proteins in eukaryotes. Supporting this notion was the description of the cell-free proteolytic system in reticulocytes,28,29 a cell that lacks lysosomes, which indicates that energy is probably required directly for the proteolytic process, although here, too, the underlying mechanisms had remained enigmatic at the time. Yet, the description of the cell-free system paved the road Inhibitors,research,lifescience,medical for detailed selleck screening library dissection of the underlying mechanisms

involved. THE UBIQUITIN-PROTEASOME SYSTEM The cell-free proteolytic system from reticulocytes28,29 turned Inhibitors,research,lifescience,medical out to be an important and rich source for the purification and characterization of the enzymes that are involved in the ubiquitin-proteasome system. Initial fractionation of the crude reticulocyte cell extract on the anion exchange resin diethylaminoethyl cellulose yielded two fractions which were both required to reconstitute the energy-dependent proteolytic activity that is found in the crude extract: the unadsorbed, flow-through material was denoted fraction I, and the Inhibitors,research,lifescience,medical high-salt eluate of the adsorbed proteins was denoted fraction II (Table 1).38 This was an important

observation and a lesson for the future dissection of the system. For one it suggested that the system was not composed of a single “classical” protease that has evolved evolutionarily to acquire energy dependence (although such energy-dependent proteases, Inhibitors,research,lifescience,medical the mammalian 26S proteasome Inhibitors,research,lifescience,medical (see below) and the prokaryotic Lon gene product have been described later) but that it was made of at least

two components. This finding of a two-component, energy-dependent protease left the researchers with no paradigm to follow, and, in attempts to explain the finding, they suggested, for example, that the two fractions could represent an inhibited protease and its activator. Second, learning from this reconstitution experiment and the essential dependence between the two active components, we continued to reconstitute activity from resolved fractions whenever we encountered Drug_discovery a loss of activity along further purification steps. This biochemical “complementation” approach resulted in the discovery of additional enzymes of the system, all required to be present in the reaction mixture in order to catalyze the multistep http://www.selleckchem.com/products/Imatinib(STI571).html proteolysis of the target substrate. We chose first to purify the active component from fraction I. It was found to be a small, ~8.5 kDa, heat-stable protein that was designated ATP-dependent proteolysis factor 1 (APF-1).