KINETIC PROPERTIES OF RAT LIVER CYTOCHROME C OXIDASE:EFFECTS INDUCED BY DIETARY CYNIDE INTOXICATION AND PROTEIN DEPLETION

Abstract

Preliminary studies on the energy-transducing capacity of the hepatic mitochondria of protein-depleted animals (obtained by feeding 3.47% dietary protein for 10 weeks) showed decrease of at least (i) 31% of the ADP-stimulated (metabolic state 3) respiration (ii) 40% of respiratory control ratios and (iii) 24.5% of ADPTO ratios when either β-hydroxybutyrate or succinate was used as substrate. Inclusion of cyanide in the low-protein diet further reduced these parameters by at most 25%. Purified and spectrally characterized cytochrome o oxide was obtained from the liver mitochrondia of animals fed with the various control (16.7%protein) and experimental (3.47% protein) diets which contained varying amounts of cyanide. The specific activities of the membrane-bound enzymes was reduced by 35% during protein depletion: addition of cyanide raised the extent of reduction to 48% at 500prm cyanide. Although the specific activity of the purified enzyme from the protein-depleted animals was 91% lower than that of control animals fed with high-protein diet only, inclusion of cyanide to this diet seemed to have greater effect (about 39% at 500 ppm CN) on the specific activity of the enzyme assessed by the use Eadie–Hofstee plot, revealed that incorporation of cyanide into the high-protein diet caused the Ka to increase from 3.16uM for control animals(on high-protein diet only) to 5.69uM and 9.95uM respectively for animals placed on high-protein diet plus 350ppm and 500ppm inorganic cyanide. Conversely, Vmax values were decreased by at most 49.5%. While the Km values of the membrane-bound enzyme from protein-depleted rats were elevated by at least 6-fold and at most by 10-fold in the mitochondria of animals fed with low-protein and 500ppm cyanide, respectively. Vmax values were elevated by 3-fold in protein-depleted animals and by 6-fold in animals fed with low-protein plus 500ppm cyanide. Lin-weaver-Burk plots of the purified cytochrome o oxidase revealed increases by 2½-, 4-, 4½-, and 9 folds in Km values in animals fed with low-protein, 200ppm, 350ppm and 500ppm inorganic cyanide respectively. Vmax values on the other hand were reduced by at least 88% in protein-depleted animals whether or not cyanide was included in the diet. Further studies on the inhibition of purified liver cytochrome o oxidase in vitro showed that cyanide is a competitive inhibitor of the enzyme with Ki values of 1.78Um at 5Um cyanide, and 2.00Um at 10Um cyanide. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) of the mitochondria revealed that while about 35 different polypeptides were present in the mitochondria isolated from animals fed with high-protein diet with or without cyanide, fewer polypeptides (24) were observed in patterns of mitochondria obtained from animals placed on low-protein diet with or without cyanide. Furthermore, the SDS-PAGE of purified liver cytochrome o oxidase showed eight subunits in the enzyme from control animals, while only the first five subunits were observed in the pattern obtained fromprotein depleted animals. These findings demonstrate unequivocally that the energy-transducing capacity of mitochondria is impaired during protein depletion as a result of the inability of the cell and mitochondrion to synthesis and insert certain polypeptides of the Nitchell’s o/r loops, most especially cytochrome o oxidase whose kinetics features are altered, irrespective of cyanide intoxisation.