ANALOGS OF RIBOFLAVIN AND FAD 557 



to establish a true uncoupling action, although by no means so specific as 

 with DNP since Og uptake is usually reduced simultaneously with the 

 P : ratio. 



Baltscheffsky (1960 b) found that light-induced phosphorylation in spin- 

 ach chloroplasts is strongly inhibited by quinacrine, 0.04 mM producing 

 almost complete block, and in cell-free extracts of Rhodospirillum rubrum 

 less potently (Baltscheffsky and Baltscheffsky, 1958; Baltscheffsky, 1960 a). 

 The inhibition is reversed by FMN and FAD in the the bacterial extracts, 

 but not at all in the chloroplasts; indeed, in the latter FMN and FAD are 

 quite potent inhibitors. It was suggested that an endogenous flavin is a 

 necessary component of the system. Photophosphorylation has recently been 

 found to be very sensitive to quinacrine. In Rhodospirillum chromatophores 

 quinacrine begins to depress the photophosphorylation at 0.0001 mM, in- 

 hibits 65% at 0.028 mM, and blocks completely at 0.1 mM, the K, being 

 0.003 mM (Horio and Kamen, 1962 a). The characteristic response to ribo- 

 flavin is, however, not prevented and it was postulated that quinacrine 

 binds at some locus in the respiratory chain. Quinacrine at concentrations 

 around 0.05 mM uncouples all types of photophosphorylation in Swiss chard 

 chloroplasts and simultaneously stimulates the photoreduction of dichloro- 

 phenolindophenol (Gromet-Elhanen and Avron, 1963). Similar effects were 

 observed in spinach chloroplasts, quinacrine at 0.02 mM inhibiting photo- 

 phosphorylation 61% and at 0.05 mM inhibiting completely, at the same 

 time stimulating the photoreduction of trimethyl-l,4-benzoquinone (Dilley 

 and Vernon, 1964). Changes in light absorption and scattering indicate a 

 relationship between photosphosphorylation and structural alterations in 

 the chloroplasts, but it is not known if quinacrine modifies directly these 

 structural changes. 



The determination of the inhibitor constant, K„ for quinacrine and sim- 

 ilar substances is somewhat more complex than with most inhibitors, due 

 to the fact that equilibrium is difficult to achieve and mutual depletion 

 kinetics must be applied, the free concentrations of both quinacrine and 

 FAD being much lower than the total concentration. HeUerman et al. (1946) 

 considered these problems relative to the inhibition of D-amino acid oxidase 

 and described a very useful technique with the appropriate equations for 

 the calculation of K^, The ^fad is 0.00057 mM, and K^ for quinine is 

 0.67 mM (means for two enzyme preparations). The quinine inhibition is 

 quite competitive but quinacrine behaves atypically and its K^ varies with 

 the experimental conditions (it is somewhat smaller than the K^ for qui- 

 nine). 



An interesting example of the effects of pH on inhibition was reported 

 by Molinari and Lara (1960) for the lactate dehydrogenase of Propionihac- 

 terium pentosaceum (Fig. 2-19). Increase of pH augments the inhibition by 

 quinacrine whereas the opposite effect is seen on Dicumarol inhibition, 



