VOL. 12 (1953) ENZYMIC OXIDATION OF /3-HYDROXYBUTYRATE 1 93 



The necessity for "sparking" (which is also required for oxidation of saturated fatty 

 acids^^) may thus be taken as an indication that oxidative phosphorylation processes 

 may be involved in "activating" the (i-isomer of j8-hydroxybutyric in some manner so 

 that it can undergo oxidation to acetoacetate and possibly also citrate. It may be con- 

 cluded from the foregoing experiments that both the d- and /-isomers of ^-hydroxy- 

 butyric acid undergo biological oxidation via the tricarboxylic acid cycle in kidney 

 preparations, but that the initial stages of oxidation of the two isomers are quite 

 different in liver mitochondrial preparations. 



Dialyzed aqueous extracts of acetone-dried rat liver mitochondrial preparations 

 were found to catalyze the reduction of diphosphopyridine nucleotide (DPN+) at the 

 expense of d- and /-BOH under appropriate experimental conditions. Study of these 

 extracts permitted delineation of the essentially different mechanisms involved. The 

 course of these reactions was followed by measuring changes in optical density at 340 m^u,, 

 caused by appearance or disappearance of the reduced form of the nucleotide, by the 

 now widely used spectrophotometric technique originated by Prof. Otto Warburg. 



TABLE IV 



"sparking" of oxidation of ^-^-hydroxybutyrate 



All vessels contained 0.005 ^ MgClj, o.ooi M ATP, 0.014 ^^ phosphate buffer pH 7.5, 0.06 M 

 KCl and washed particles from 0.45 g rat liver. Total volume 3.0 ml. Concentration of d-HOH where 

 added was o.oi M. Fumarate was added in concentrations shown. In experiment I, Temperature 

 22° C, time 65 minutes. In experiment II, temperature 20° C, time 75 minutes. 



,- . , c 1 , , Fumarate Oo uptake , ,. 



Expt. Substrate ... " , formation 



concentration microatoms ^ 



A cetoacetate 

 formation 

 micromoles 



1-3 

 0.1 

 2.6 

 0.0 



4.0 

 II none none 1.8 0.3 



0.7 

 0.2 



3-4 

 0.3 



5-6 



0.3 

 1.9 



0.3 

 0.2 



Such extracts contain the already known^, DPN-linked /-specific /S-hydroxy- 

 butyric dehydrogenase, as is shown by the data in Table V. There is virtualty no reduc- 

 tion of DPN+ in the absence of added substrate or with d-BOH as substrate. It is seen 

 that this reaction requires only extract, /-BOH, DPN+ and cysteine (/S-hydroxybutyric 

 deh57drogenase is known to be dependent on -SH groups-^'^^) and addition of ATP, Mg++ 

 and CoA does not affect the reaction. It is concluded that the oxidation of /-BOH in 

 these extracts occurs by the reaction 



/-/S-hydroxybutyrate -f DPN+ ^^ acetoacetate + DPNH + H+ (2) 



In similar experiments it was found that acetoacetate is reduced by DPNH, as would be 

 expected, since reaction (2) is known to be readily reversible. 



References p. 202. 



