142 



M. E. JONES, S. BLACK, R. M. FLYNN, F. LIPMANN 



VOL. 12 (1953) 



autolyzes rather quickly. To promote this further, 33 ml of M dipotassium phosphate are mixed witli 

 1,000 g of yeast and the mixture stirred overnight in the cold room at 0-5°. The yeast autolysate is 

 centrifuged in a cold centrifuge. The supernatant solution does not store well and it is desirable to 

 carry it immediately through the first ammonium sulfate fractionation step. 



Sonic prcpavations. One part of fresh Bakers' yeast is mixed to a paste with i part of o.i 71/ 

 cold dipotassium phosphate and exposed to vibration in a 10 kilocycle magnetostrictive oscillator 

 (Ra3d;heon Manufacturing Company) for 40 minutes. The mixture is centrifuged in the cold in a 

 Servall centrifuge at top speed (12,000 r.p.m.) for 30 minutes. The supernatant solution, amounting 

 to slightly more than half the original volume, may be frozen and kept for a considerable length of 

 time in the deep freeze. 



This extract has the same specific activity as fraction i in Table I but is only half as concentrated. 

 It behaves on fractionation like the ether extract and, after fractionation step 3, the two preparations 

 become very similar in all respects. 



Protamine precipitation. The slightly turbid supernatant solution is treated with protamine. The 

 precipitate, containing very little active material, is discarded. For every mg of nucleic acid, 0.5 mg 

 of protamine is added, using a 2% protamine sulfate solution. The protamine precipitate is centrifuged 

 at 0° in a cooled centrifuge. 



The nucleic acid content is determined turbidimetrically in the following manner: to 0.05 ml 

 of the solution to be analyzed are added 4.5 ml of 0.05 M phosphate buffer, pH 6.1, and 0.5 ml of 

 2% protamine sulfate solution. The tubes are mixed and the turbidity is measured on a Klett colori- 

 meter using filter No. 54. The reading is compared with a standard curve prepared with o.i to 0.6 mg 

 of yeast nucleate adjusted to pH 6. 



First ammonium sulfate precipitation. To 1,000 ml of clear supernatant solution (pH about 6). 

 350 g of soHd ammonium sulfate are added (55% saturation). The active material is precipitated and 

 centrifuged off; the supernatant solution is discarded. The precipitate is dissolved with 66 ml of 

 0.05 M potassium bicarbonate solution. The resulting solution is 20% saturated with ammonium 

 sulfate. 



Second ammonium sulfate precipitation. Enough saturated ammonium sulfate solution is added 

 to bring the above enzyme preparation from 20% to 35% saturation with respect to ammonium 

 sulfate. The precipitate is discarded and the supernatant solution is brought to 45% saturation with 

 saturated ammonium sulfate solution. The precipitate is dissolved in 0.05 M bicarbonate solution and 

 is generally used in this form without further treatment. The enzyme is stable in ammonium sulfate 

 for a considerable length of time when stored in the deep freeze, while dialyzed enzyme preparations 

 are much less stable even when frozen. Therefore, in most cases, dialysis is omitted. However, for 

 certain experiments, it is desirable to remove the ammonium sulfate. For this purpose, the solution 

 may be dialyzed with agitation at 0° against a solution of 0.05 M potassium bicarbonate in 0.5% 

 potassium chloride for 2 to 3 hours. Typical results of the above fractionation procedure are illustrated 

 in Table I. 



TABLE I 



ACTIVITY OF VARIOUS YEAST FRACTIONS 



* Turbidimetric^. 



Assay system; enzyme units. In general, enzyme activity was determined by the hydroxamic acid 

 method as described by ChouI^. Chou observed that, for the assay of the isolated ATP-Co.\-acetate 

 donor system, contained in the 40% acetone precipitate from pigeon liver extract (A-40 fraction), 

 high concentrations of hydroxylamine were necessary. He also found that, in the pigeon liver fraction 

 precii)itating between 40% and 60% acetone (A-60 fraction) which contained the acceptor enzyme 

 for aromatic amines, there was present another enzyme which catalyzed the reaction of acetyl CoA 

 with hydroxylamine. The presence of the latter enzyme was demonstrated by the fact that, if the 

 A-40 and A-60 fractions were combined, hydroxamic acid formation was maximal at low concentra- 

 tions of hydroxylamine. The cruder yeast preparations seem also to contain an enzyme which cataly- 

 zes the hydroxylamine reaction. As shown in Chou's experiments, the effect of the hydroxylamine 



References p. 149. 



