Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Intracellular proteases are ubiquitous in nature. This extensive distribution necessarily implicates their participation in a wide variety of cellular events. Escherichia coli, contains a variety of proteases and peptidases. For the most part, the known endoproteases of E. coli have been characterized from cells grown to stationary phase on enriched media. Surprisingly, no studies have addressed the question of what proteases are present in exponentially growing E. coli, despite the fact that significant protein turnover occurs.;Previous studies from this laboratory revealed the presence of a unique Ca{dollar}\sp{lcub}2+{rcub}{dollar}-activated proteolytic activity in E. coli. This activity was maximal in mid-log grown cells but absent at stationary phase and was restricted to the periplasmic space of E. coli. The periplasmic fraction was initially analyzed by DEAE-cellulose chromatography. The observation that the periplasm contained multiple proteolytic activities is the basis for the studies reported here.;These samples were purified by conventional column chromatography techniques. This approach has resulted in the isolation of four apparently distinct proteases designated Protease peri 3, peri 4, An, and X. Proteases peri 3 and peri 4 are monomeric metalloendoproteases of 94 kDa and 102 kDa respectively. These enzymes degrade both casein and insulin B chain. Protease peri 3 is stabilized by Ca{dollar}\sp{lcub}2+{rcub}{dollar} or Mn{dollar}\sp{lcub}2+{rcub}{dollar}, cleaves insulin B chain at four positions and is immunologically distinct from other periplasmic proteases, including Protease III. Protease peri 4 cleaves insulin B chain at a single location.;Proteases An and X mainly degrade casein and were further examined due to their size ({dollar}>{dollar}3000 kDa) on gel filtration. Protease An is a metalloendoprotease activated by ATP. Protease X, however, is a serine protease which is unaffected by ATP. These two proteases exhibit a number of properties similar to the "proteasomes" observed in eukaryotic systems.;Whether each of these proteases represent a unique gene product remains to be established. It is the hope that this study will promote future work that may then primarily focus on the physiological role of these proteases in the periplasm.



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