TABLE 1.

Purification of single-U addition activity

Fractiona	Total protein (μg)	n+1 Ub			Ligasec
		Total activity (counts)d	Sp act (counts/μg)	Purification (fold)	Total activity (counts)	Sp act (counts/μg)	Purification (fold)
Mitochondria	90,644	213,742	2.36	NAe	1,054,606	11.63	NA
Heparin-Sepharose	9,743	171,139	17.56	7.45	3,400,831	349.04	30.0
Q-Sepharose	960	209,049	217.67	92.31	3,509,089	3,653.82	314.05
Glycerol gradient	140	374,897	2,677.84	1,135.62	1,502,781	10,734.15	922.61

^aThe mitochondrial fraction is the clarified (20,000 × g for 15 min) supernatant of the 0.5% Triton X-100-solubilized mitochondrial lysate described in Materials and Methods. The heparin-Sepharose, Q-Sepharose, and glycerol gradient fractions correspond to the pooled peaks of single-U addition and ligase activities from each chromatography step. 

^bn+1 U refers to single-U addition, as assayed by single-U extension on a poly(A)₂₀ RNA substrate. 

^cligase refers to the quantitation of adenylated 57-kDa ligase. 

^dCounts are arbitrary units quantitated from an Applied Biosystems phosphorimager with the background subtracted. 

^eNA, not applicable.