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.

Photoaffinity labeling the torpedo nicotinic acetylcholine receptor with [(3)H]tetracaine, a nondesensitizing noncompetitive antagonist.

Tetracaine (N,N-dimethylaminoethyl-4-butylaminobenzoate) and related N,N-dialkylaminoethyl substituted benzoic acid esters have been used to characterize the high-affinity binding site for aromatic amine noncompetitive antagonists in the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Tetracaine binds at equilibrium to a single site with a K(eq) value of 0.5 microM in the absence of agonist or presence of alpha-bungarotoxin and with a K(eq) value of 30 microM in the presence of agonist (i.e., for nAChR in the desensitized state). Preferential binding to nAChR in the absence of agonist is also seen for N,N-DEAE and N,N-diethylaminopropyl esters, both binding with 10-fold higher affinity in the absence of agonist than in the presence, and for the 4-ethoxybenzoic acid ester of N, N-diethylaminoethanol, but not for the 4-amino benzoate ester (procaine). Irradiation at 302 nm of nAChR-rich membranes equilibrated with [(3)H]tetracaine resulted in covalent incorporation with similar efficiency into nAChR alpha, beta, gamma, and delta subunits. The pharmacological specificity of nAChR subunit photolabeling as well as its dependence on [(3)H]tetracaine concentration establish that the observed photolabeling is at the high-affinity [(3)H]tetracaine-binding site. Within alpha subunit, >/=95% of specific photolabeling was contained within a 20-kilodalton proteolytic fragment beginning at Ser(173) that contains the M1 to M3 hydrophobic segments. With all four subunits contributing to [(3)H]tetracaine site, the site in the closed channel state of the nAChR is most likely within the central ion channel domain.