376 B. MAGASANIK 



isolation of a crystalline PNA-protein from carp muscle.^* This compound, 

 nucleotropomyosin, is the first crystalline nucleoprotein to be prepared 

 from animal tissue and possesses considerable chemical and biological 

 interest. Together with nucleotropomyosin, another crystalline protein, the 

 well-known muscle component tropomyosin, was obtained. The two pro- 

 teins were indistinguishable by electrophoresis but behaved differently in 

 the ultracentrifuge. Tropomyosin was free from PNA and identical with 

 the protein portion of nucleotropomyosin. In contrast to the complex 

 cytoplasmic nucleoproteins isolated so far, nucleotropomyosin appears to 

 be free of phospholipids. The method of preparation is given in detail. 



(1) Nucleotropomyosin from Carp Muscle.^^ The preparation is carried out through- 

 out in the cold and all separations are done by centrifugation. Carp muscles cut 

 with a freezing microtome into slices 40 fi thick are extracted for 20 minutes with 

 3 vol. of KCl-phosphate solution (0.50 M KCl and 0.1 M KH2PO4 brought to pH 5.). 

 This extract is diluted with 3.5 vol. of cold water; the precipitate is discarded and the 

 supernatant (I) kept. 



The residue from the first extraction is reextracted for 10 minutes with 3 vol. of 

 0.5 M phosphate solution of pH 6.5 containing 0.3% sodium adenosinetriphosphate. 

 The residue is discarded and the extract (II) is diluted with 7 vol. of water. A pre- 

 cipitate of myosin and nucleotropomyosin forms, which is washed twice with water 

 and redissolved in 0.5 M KCl at neutral pH. The supernatant II and the supernatant 

 I are mixed and brought to 4.6. The precipitate containing tropomyosin is washed 

 twice with water and redissolved in 0.5 M KCl at neutral pH. 



Both solutions are now centrifuged for 30 minutes at 14,000 r.p.m. to remove some 

 turbid material and are purified by a second precipitation by dilution with 8 vol. of 

 water at neutral pH (nucleotropomyosin) or at pH 4.6 (tropomyosin). Both precipi- 

 tates are washed twice with water and redissolved in 0.5 M KCl at neutral pH. 



Both tropomyosins are isolated from these two solutions by (NH4)2S04 fractiona- 

 tion at neutral pH: the major part of the total protein content of the solutions pre- 

 cipitates between 30 and 50% saturation, while the tropomyosins precipitate between 

 50 and 66% saturation. The precipitate can be redissolved quickly by a slight dilu- 

 tion with water, giving a water-clear solution. 



Although these methods of preparation are very reproducible, some variations are 

 observed in the yields obtained, which are usually 0.07% of the wet weight for nucleo- 

 tropomyosin and 0.03% for tropomyosin. 



The undiluted salted-out precipitates are used for crystallization. This is carried 

 out by dialyzing an appro.ximate 1.5% solution against a solution containing 16 g. 

 (NH4)2S04 per liter and 0.01 M acetate buffer of pH 5.4. Nucleotropomyosin crystal- 

 lizes in elongated prisms; tropomyosin in the quadrangular plates previously de- 

 scribed by Bailey.'^ 



3. Isolation of Nucleoprotein from the Tissues of Higher Plants 



a. Fractional Centrifugation 



This method has been used extensively for the isolation of plant viruses 

 from leaves. The mildness of the procedure permits the isolation of some 



" G. Hamoir, Biochem. J. 48, 146 (1951). 

 !•* K. Bailey, Biochem. J. 43, 271 (1948). 



