Kinetic evidences for facilitation of peptide channelling by the proteasome activator PA28

被引:54
作者
Stohwasser, R
Salzmann, U
Giesebrecht, J
Kloetzel, PM
Holzhütter, HG
机构
[1] Humboldt Univ, Med Fak Charite, Inst Biochem, D-10117 Berlin, Germany
[2] Deutsch Inst Ernahrungsforsch, Abt Pravent Med Lebensmittelforsch, Bergholz Rehbrucke, Germany
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2000年 / 267卷 / 20期
关键词
20S proteasome; fluorogenic peptides; kinetic modelling; PA28; activator;
D O I
10.1046/j.1432-1327.2000.01706.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The activation kinetics of constitutive and IFN gamma-stimulated 20S proteasomes obtained with homomeric (recPA28 alpha, recPA28 beta) and heteromeric (recPA28 alpha beta) forms of recombinant 11S regulator PA28 was analysed by means of kinetic modelling. The activation curves obtained with increasing concentrations of the individual PA28 subunits (RecP28 alpha/RecP28 beta/RecP28 alpha + RecP28 beta) exhibit biphasic characteristics which can be attributed to a low-level activation by PA28 monomers and full proteasome activation by assembled activator complexes. The dissociation constants do not reveal significant differences between the constitutive and the immunoproteasome. Intriguingly, the affinity of the proteasome towards the recPA28 alpha beta complex is about two orders of magnitude higher than towards the homomeric PA28 alpha and PA28 beta complexes. Striking similarities can been revealed in the way how PA28 mediates the kinetics of latent proteasomes with respect to three different fluorogenic peptides probing the chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing like activity: (a) positive cooperativity disappears as indicated by a lack of sigmoid initial parts of the kinetic curves, (b) substrate affinity is increased, whereby (c), the maximal activity remains virtually constant. As these kinetic features are independent of the peptide substrates, we conclude that PA28 exerts its activating influence on the proteasome by enhancing the uptake (and release) of shorter peptides.
引用
收藏
页码:6221 / 6230
页数:10
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