TABLE 3.

Specificities of Ac-PRQR-al and Ac-EPFD-al as proteasome inhibitors

Reaction mixtures (50 μl) contained 0.32 μg of 20 S proteasome, 1 μg of PA28γ (K188E), and either reticulocyte buffer alone or 2 μm Ac-PRQR-al, 2 μm Ac-EPFD-al, or 20 μm lactacystin. After incubation at 37 °C for 30 min, 50 μl of a 200 μm solution of the indicated fluorogenic peptide was added to initiate degradation. Duplicate samples were quenched with 200 μl of ethanol at 10 and 30 min, and fluorescence was measured at ex/em 380/440 nm for MCA substrates and ex/em 335/410 for the βNA substrate Cbz-LLE. The measured fluorescence was divided by the fluorescence obtained in the absence of inhibitor to produce % inhibition. A negative value indicates stimulation of peptide hydrolysis in the presence of the inhibitor. Note that whereas lactacystin markedly inhibited hydrolysis of sLLVY-MCA (a substrate for the chymotrypsin-like activity of the proteasome) and bLRR-MCA (a substrate for the trypsin-like subunit), the peptide aldehydes are strikingly specific for just one proteasome active site.

Fluorogenic peptide	% Inhibition
	Ac-PRQR-al	Ac-EPFD-al	Lactacystin
Suc-LLVY-MCA	-32	-5	97
Boc-LRR-MCA	97	-22	63
Cbz-LLE-βNA	2	93	10