Seol J H, Yoo S J, Shin D H, Shim Y K, Kang M S, Goldberg A L, Chung C H
Department of Molecular Biology and Research Center for Cell Differentiation, College of Natural Sciences, Seoul National University, Korea.
Eur J Biochem. 1997 Aug 1;247(3):1143-50. doi: 10.1111/j.1432-1033.1997.01143.x.
HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.
大肠杆菌中的HslVU是一种新的双组分ATP依赖性蛋白酶，由两种热休克蛋白组成，即HslU ATP酶和与蛋白酶体β型亚基相关的HslV肽酶。我们在此表明，重组后的HslVU酶不仅能降解某些疏水肽，还能降解各种多肽，包括胰岛素B链、酪蛋白和羧甲基化乳白蛋白。当HslV（一种250 kDa的复合物）与HslU（一种450 kDa的复合物）的摩尔比为1:2时，可获得最大蛋白水解活性。单独的HslV能缓慢水解这些多肽，但其活性在ATP存在下被HslU刺激了20倍。蛋白底物可将HslU的ATP酶活性刺激高达50%，但非水解蛋白则不能，并且在HslV存在下这种刺激进一步增加了2至3倍。能最大程度刺激ATP酶的胰岛素B链浓度允许B链水解的最大速率。此外，添加越来越多的ADP或N - 乙基马来酰亚胺会同时降低ATP以及蛋白质或肽的水解。因此，HslVU既是一种蛋白激活的ATP酶，也是一种ATP依赖性蛋白酶，并且这些过程似乎是相关联的。令人惊讶的是，蛋白质和肽底物在水解方面并不相互竞争。乳胞素强烈抑制蛋白质降解，但对肽水解影响很小，而肽醛是小肽水解的有效抑制剂，但对蛋白质影响很小。因此，肽和蛋白质的ATP依赖性水解的功能需求似乎不同。
