TABLE 3.

Effect of carboxypeptidase treatment on activities of PA700 and PA700 subcomplexes

The indicated purified protein complexes were assayed for ATPase activity or 20 S proteasome activating activity after preincubation in the absence (−) or presence (+) of CbpA. CbpA treatment was conducted either after (row 2) or before (row 3) preincubation of PS-1, PS-2, and PS-3 (4 μg each) for reconstitution. ATPase activity was determined by the Malachite green method. Proteasome activating activity represents the relative increase of 20 S proteasome activity (measured by the rate of hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC) by the indicated proteins. The activity of 20 S proteasome (0.4 μg/assay) in the absence of other proteins is set to a relative value of 1.0. Data represent mean values of triplicate assays ± S.E. Similar results were observed in at least three independent experiments for each condition and in one experiment using Suc-Leu-Leu-Glu-AMC as substrate.

Protein	ATPase activity		Proteasome activating activity (relative activity)
	−CbpA	+CbpA	−CbpA	+CbpA
	pmol Pi/min/μg protein
PA700	28.8 ± 3.6	26.9 ± 3.4	22.8 + 0.7	1.2 ± 0.5
(PS-1/PS-2/PS-3) ± CbpA	18.9 ± 2.8	15.3 ± 3.9	17.2 ± 1.6	1.3 ± 0.8
CbpA ± (PS-1/PS-2/PS-3)	20.5 ± 2.0	18.6 ± 1.5	16.0 ± 3.2	1.3 ± 0.2