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Table I

Purification of Bioactivity from Pig Brain

FractionVolumeProteinProteinSpec. Act.ActivityPurificationYield
  ml  mg/ml  mg  U/mg  U  fold  % 
Pig brain 100k supernatant  3,000   6.4  19,000      0.8  15,400   100 
Q-Sepharose step eluate    150  10   1,500     35  52,500       44 (44)  340 (100) 
Blue 3GA–Sepharose     25   3.8      95    200  19,000     250 (6)  125 (36) 
Phenyl–Sepharose      7   1.3       9  1,200  11,000       1,500 (6)   73 (21) 
Q-Sepharose      3   0.15       0.45  8,000   3,600  10,000 (6.7)   23 (7) 
FractionVolumeProteinProteinSpec. Act.ActivityPurificationYield
  ml  mg/ml  mg  U/mg  U  fold  % 
Pig brain 100k supernatant  3,000   6.4  19,000      0.8  15,400   100 
Q-Sepharose step eluate    150  10   1,500     35  52,500       44 (44)  340 (100) 
Blue 3GA–Sepharose     25   3.8      95    200  19,000     250 (6)  125 (36) 
Phenyl–Sepharose      7   1.3       9  1,200  11,000       1,500 (6)   73 (21) 
Q-Sepharose      3   0.15       0.45  8,000   3,600  10,000 (6.7)   23 (7) 

3 liters of high speed supernatant derived from pig brain homogenate was fractionated by successive passage over Q-Sepharose, Blue 3GA–agarose, phenyl–sepharose, and Q-Sepharose. In each case, column fractions were assayed for competence to restore a GTPγS-dependent biological response in permeabilized cells. The peaks of activity were pooled; the specific activities shown in this table are calculated as described in Materials and Methods and representative of at least three determinations. Parenthetical figures for purification and yield show change in those figures from the previous stage of purification. The fractions obtained after gel filtration were too dilute for accurate protein assay; therefore, since specific activities could not be determined, relative activities were assessed per volume of protein added, and these activities are listed separately in Fig. 6.  

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