ANALOGS OF NICOTINAMIDE 485 



thesis and breakdown are shown in the accompanying diagram. The major 

 route of NAD formation, at least in mammalian tissues, is probably through 

 reactions (l)-(3) since the alternative pathway (8)-(10) is kinetically and 

 thermodynamically unfavorable. Analogs of nicotinate can thus either di- 

 rectly inhibit any of these reactions or enter into the reactions to form 

 abnormal intermediates, and perhaps analogs of NAD or NADP, which 

 are inhibitory. (See reactions on page 486). 



Inhibition of NAD Nucleosidase (NADase) by Nicotinamide 

 and Related Compounds 



It will be convenient to discuss first the direct inhibitions by simple 

 pyridine derivatives and then proceed to those substances incorporated into 

 NAD analogs. There is a constant turnover of NAD in tissues and at least 

 a fraction of the degradative process is attributable to NADase, and in 

 tissue extracts or homogenates the splitting of NAD may be an important 

 factor determining the dehydrogenase activity. Thus inhibitors of NADase 

 might be expected under certain circumstances to protect the coenzyme. 

 Furthermore, it will be evident later that the mechanisms of NADase inhi- 

 bition are involved in the formation of abnormal NAD analogs. Mann and 

 Quastel (1941) were the first to observe an inhibition of NAD breakdown 

 by nicotinamide. They worked with brain suspensions and determined NAD 

 by adding lactate dehydrogenase and lactate. Nicotinamide at 25 mM was 

 found to prevent the breakdown of NAD almost completely, and addition 

 of nicotinamide increases the respiration of various systems oxidizing lactate 

 by preventing the destruction of NAD. Nicotinate, on the other hand, is 

 completely inactive. Many investigators have subsequently used nicotin- 

 amide to preserve NAD in various preparations, often in very high concen- 

 trations and without regard for the other possible inhibitions it might exert. 

 Handler and Klein (1942) soon showed that NADP splitting is also inhib- 

 ited by nicotinamide. 



Mcllwain and Rodnight (1949) pointed out that the indiscriminate use 

 of high nicotinamide concentrations to protect NAD in metabolic studies 

 is unnecessary, since almost complete inhibition of NADase is seen at con- 

 centrations from 2 to 10 mM (actually they showed that 2.67 mM inhibits 

 73%). The problem of the proper concentration of nicotinamide to use is a 

 difficult one because the NADases of various tissues and organisms show 

 marked differences in susceptibility to inhibition. The early work was all 

 done on brain NADase, which is quite sensitive, and it has been found that 

 some other NADases are also sensitive, e.g., from beef spleen (Zatman et 

 al., 1953). However, the enzymes from rabbit erythrocytes (Alivisatos and 

 Denstedt, 1952; Rubinstein et al., 1956; Malkin and Denstedt, 1956), mouse 

 mammary gland and tumor (Branster and Morton, 1956), and lupine seed- 

 lings (Hasse and Schleyer, 1961) are only moderately sensitive to nicotin- 



