ENZYMES ATTACKING NUCLEIC ACIDS 575 



is followed by a rapid deeper cleavage of the ribonuclease I accompanied 

 by a practically total loss of activity. Hirs^^'^ and Hirs, Stein, and Moore*^'' 

 applied oxidation of ribonuclease with performic acid at — 10° and subse- 

 cjuent digestion of the oxidized enzyme with trypsin to the investigation of 

 the amino acid sequence in the ribonuclease molecule. The treatment of 

 the protein with performic acid oxidized exclusively the sulfur groups of 

 the cystine and methionine components. The number of polypeptides ob- 

 tained by chromatography of the tryptic digest closely agreed with that 

 predicted from the experimentally determined values of 10 lysine and 4 

 arginine rests for each molecule of ribonuclease. 



b. Other Ribomiclcases 



The existence of ribonucleases other than the enzyme obtained in crystal- 

 lized form from pancreas can now be considered as certain. They differ 

 from pancreas ribonuclease in regard to their specificity toward the inter- 

 nucleotide bonds of the substrate, in regard to their heat lability and to 

 their pH optima. Schmidt ct a/.^^ found in 1950 that incubation of PNA 

 with crude pancreas extracts resulted in the cleavage of internucleotide 

 bonds which were resistant to the action of crystallized pancreas ribonu- 

 clease I. These bonds were the ester linkages of 3 '-purine nucleotide groups 

 with the adjacent nucleotide groups. Maver and Greco®^ reported the 

 presence in spleen extracts of a heat-labile ribonuclease which differed 

 from ribonuclease I also by the different pH range (optimum in the region 

 of pH 5.2, in the presence of Mg; at pH 6.6 without addition of Mg salts^^) 

 of its optimal activity. 



Clear evidence for the existence of different ribonucleases and for the 

 nature of their catalytic activity was obtained by Hilmoe and Heppel," 

 who fractionated the nucleolytic enzymes of spleen extracts. They studied 

 the action of these enzymes not only on PNA, but on the limit polynucleo- 

 tides obtained by exhaustive hydrolysis of PNA with ribonuclease I as 

 well as on some simple synthetic nucleotide-P-esters of well-defined struc- 

 ture. Hilmoe and Heppel prepared from spleen a ribonuclease which was 

 free from phosphomonoesterases and which hydrolyzed the limit poly- 

 nucleotide fraction of PNA approximately four times faster than PNA 

 itself. The pH optimum of this enzyme was at pH 6.6. The enzyme was 

 rapidly inactivated at 60°. 



Mechanism of Action. The spleen ribonuclease does not hydrolyze cyclic 

 nucleotides. It is capable of catalyzing exchange reactions between nucleo- 



"^ G. Schmidt, R. Cubiles, and S. J. Thannhauser, /. Cellular Coywp. Physiol 38, 



Suppl. 1,61 (1950). 

 «* M. E. Maver and A. E. Greco, /. Biol. Chem. 181, 861 (1949). 

 6« M. E. Maver and A. E. Greco, Federation Proc. 13, 261 (1954). 

 " R.. J. Hilmoe and L. A. Heppel, Federation Proc. 12, 217 (1953). 



