Studies of the ATP dependence of protein degradation in cells and cell extracts.
Experiments with metabolic inhibitors in vivo indicate that intracellular protein degradation requires the continuous production of ATP. We have established soluble cell-free preparations from rabbit reticulocytes, rat liver, and Escherichia coli that degrade abnormal protein in an ATP-dependent fashion. These enzymes appear to be responsible for the selective breakdown of abnormal protein that may result from mutations, biosynthetic errors or intracellular denaturation. Experiments with inhibitors indicate that this process and the degradation of many short-lived normal proteins does not occur in the lysosome. The cell-free extracts prepared from these crude extracts hydrolyse [14C] globin by a process stimulated 2--3-fold by ATP and to a lesser extent by GTP, CTP or UTP. These activities degrade globin to large peptides which are then cleaved by soluble peptidases. The ATP-stimulated protease that partially purified from rat liver cytoplasm is also stimulated by pyrophosphate. This protease has an apparent molecular weight of 480,000. In contrast, the E. coli enzyme has an apparent molecular weight of 115,000 and is completely dependent on ATP, after partial purification by ion exchange and gel chromatography. This enzyme can be distinguished from six other proteolytic enzymes from E. coli active at pH 7.8. E. coli contains, in addition, four proteases that are not stimulated by ATP and degrade globin to acid-soluble material. We have also demonstrated in E. coli and reticulocytes other proteases that appear specific for small protein substrates and may play a role in the later steps in protein breakdown. The ATP-stimulated endoproteases appear to catalyse the rate-limiting steps in intracellular protein breakdown. However, the actual role of ATP in the degradative process is not known.
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005914:Globins / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000201:enzymology
D011506:Proteins; D000255:Adenosine Triphosphate; D005914:Globins; D010450:Endopeptidases