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concentration would allow the fractionation of RNA polymerase away from
other mitochondrial proteins, thus effecting another purification step.
The effect of ammonium sulfate on the mitochondrial RNA synthesizing
activity was measured (Table 12) by adding to solubilized mitochondrial
RNA polymerase samples ammonium sulfate to the desired concentration with
mixing followed by centrifugation at 40,000 rpm for 90 minutes. The RNA
polymerase activity was measured in the redissolved and dialysed protein
samples as described in the methods. The results indicated that 90% of
the activity was recovered when the ammonium sulfate concentration was
50%. It was interesting to observe that when the 30,000 rpm super
(control) was centrifuged at 40,000 rpm for 70 min. 19% of the activity
was lost. This could represent the activity that is not completely
solubilized and thus sediments at 40,000 rpm but not at 30,000 rpm.
Solubilization and Purification
Of Mitochondrial RNA Polymerase
The scheme for the purification of the mitochondrial RNA polymerase
is shown in Figure 11. The optimum conditions described in Table 10
were used to solubilize mitochondrial RNA polymerase. The washed mito
chondria obtained from 182g (wet weight) of cells were purified by
treatment with pancreatic deoxyribonuclease (100 pg/ml) for thirty minutes.
The mitochondria obtained after two washings in STE buffer were suspended
in lysis buffer and either used directly for enzyme solubilization or
stored in liquid nitrogen prior to use. An equal volume of lysis mixture
was mixed with mitochondrial suspension to bring the final concentration
to 0.8% Triton X-100, 0.50 M KC1, and 0.005M EDTA. The mixture was
gently stirred for 15 minutes, 4C. The supernatant obtained after cen
trifuging at 30,000 rpm for 90 minutes was brought to 50% saturation with