NUCLEIC ACIDS AND RELATED COMPOUNDS 243 



Identification of free bases, nucleosides or nucleotides in plant extracts is basically 

 the same problem as identifying them in nucleic acid hydrolysates. Some preliminary 

 purification is probably necessary before applying unknowns to paper. Precipitation or 

 ion exchange methods as described under "Isolation" can be used for this purpose. The 

 reader is referred to papers of Rowan (33, 34) and Cherry and Hageman (35) for methods 

 suitable for detection of free nucleotides in plant extracts. 



Detailed characterization of many nucleic acid derivatives has rested heavily on 

 the action of specific enzymes on purified materials. Thus an enzyme has been prepared 

 from germinating barley {Hordeimi spp. ) or rye grass (Lolium spp. ) which specifically 

 hydrolyzes nucleotide 3' -phosphates (36). Application of this and other enzymes to struc- 

 ture determinations may be found in the general references. 



Absorption spectra are also useful for identification of purified compounds. All 

 purine and pyrimidine derivatives show strong absorption at about 260 m/i, but shifts in 

 absorption occur with changes in pH. These shifts are characteristic for specific purines 

 and pyrimidines since they depend on the ionizable groups present. In many cases enough 

 material can be extracted from a paper chromatogram to permit its identification by 

 measuring spectra at different pH values. Further discussion and presentation of spectra 

 may be found in the general references as well as references (37, 38). DPN (NAD), TPN 

 (NADP) and riboflavine derivatives have spectral properties useful in characterizing them. 

 The first two have identical spectra with a peak at about 260 mju; but more important is 

 the appearance of a peak at 340 m.\i upon reduction with sodium dithionite or with appro- 

 priate enzymes and substrates. Riboflavine derivatives show a strong yellow -green flu- 

 orescence when illuminated with light of about 445 m^. 



Qualitative and quantitative analysis for the nucleotides which function as cofactors 

 in enzyme systems may be carried out by setting up the enzyme system without the re- 

 quired cofactor, adding unknown material, and measuring enzyme activity. 



METABOLIC PATHWAYS 



The biosynthetic pathways leading to formation of nucleic acid derivatives have been 

 well clarified in bacteria and avian liver. In contrast, very few studies have been made 

 concerning metabolic pathways of these compounds in higher plants. The sequences 

 shown in Figure 1, therefore, may be considered as probable but not confirmed for higher 

 plants. Reviews of this area are regularly presented in The Annual Review of Biochem- 

 istry (e.g., 39). 



In addition to compounds directly related to the nucleic acids, compounds of similar 

 structure have been considered in this chapter since they are probably synthesized along 

 somewhat similar pathways. Riboflavin, for instance, at least in the microorganism 

 Eremothecimn ashbyii, seems to be synthesized from various purines (40). Guanine is 

 the best precursor. Tracer studies of Krupka and Towers (41) have shown glycine to be 

 a good precursor of allantoin in wheat as would be expected if the indicated scheme of 

 purine synthesis were followed. The methyl groups of thymine and the methylated xan- 

 thines has received some attention and probably fit into the usual scheme of biological 

 methylations involving formate and methionine. The synthesis of caffeine in Coffea 

 arabica has been indicated by tracer experiments to follow the normal purine pathways 

 with methyl groups coming from methionine (42). 



The most important generalization which has emerged from studies of the sequence 

 is that transformations of the nitrogen bases are always carried out not on the free bases 

 but on nucleotide derivatives. In chicken embryos it is likely that ribonucleotides are 



