Liver isotope exchange

This mechanism in which reactant coenzyme may leak from the ternary complex was first suggested for liver alcohol dehydrogenase to reconcile the results of initial rate studies with primary and secondary alcohols as substrates and isotope exchange experiments (39), and it has also been proposed for yeast alcohol dehydrogenase (40) and a-glycero-phosphate dehydrogenase from rabbit muscle ( l). 

The conclusion that even with the best substrate, ethanol, dissociation of NAD occurs from the active ternary complex is consistent with the evidence-from isotope exchange experiments 32), mentioned previously, that the dissociation of coenzyme is not greatly suppressed in the ternary complex compared with the binary complex, in contrast to liver alcohol dehydrogenase. This is also indicated by the initial rate data in another way 4>a/4>o for the preferred pathway mechanism approximates to the dissociation constant for NAD from the ternary complex (Table I), and is reasonably constant for the three primary alcohols and approximately equal to the dissociation constant of E-NAD, determined independently 40). 

Mehler and Tabor B48) performed a particularly careful experiment to demonstrate that urocanic acid is an intermediate in the catabolism of histidine in the liver. This was done by incubating histidine-2-C with crude liver preparations in the presence of twenty times the histidine concentration of unlabeled urocanic acid. From samples taken at zero time and at the end of the incubation period it was estimated that most of the radioactivity disappearing from the histidine appeared as urocanic acid. To rule out the possibility that the C in the urocanic acid was derived by some exchange reaction, while net degradation of histidine could occur by a different reaction, an experiment was performed with unlabeled histidine and C -urocanate. The isolated histidine was isotope-free. This result demonstrated that the conversion of histidine to urocanate is not reversible and that there is no chemical exchange reaction of an unknown nature. 

The evidence supporting this concept is derived from experiments on yeast extracts and pigeon liver preparations but further joint studies by Lipmann and Lynen make the formation of phosphoryl-ated or pyrophosphorylated coenzyme A improbable and suggest the formation of enzyme-bound adenosine phosphate and coenzyme A. In the presence of purified yeast enzymes isotopic pyrophosphate was found to exchange the isotope with ATP in the absence of coenzyme A. This excludes the latter as an obligatory participant. Isotopic acetate also exchanged readily with acetyl coenzyme A in the presence of this yeast enzyme. The following scheme of 3 reactions fits the facts ... 

Newly synthesized plasma cholesterol, predominantly of hepatic origin, rapidly achieves equilibrium in vivo with the cholesterol in the stroma of the erythrocyte. This same equilibrium between the plasma free cholesterol and that of the red cell has been demonstrated in vitro, 50% equilibration took place in 1 hour. Ester cholesterol did not participate in this exchange. The equilibration probably is brought about by an exchange reaction at the several interfaces. An exchange of a similar nature could account for the relatively uniform distribution of isotopic cholesterol in liver, plasma, and red cells after acetate-C administration and for the rapidity with which this uniformity is achieved. 

The measurements of N enrichment have been performed on DNA isolated from chicken embryos liver. We have chosen chicken embryos because they develop in a closed system without endogenous nutrients and water supply and thus nitrogen metabolism is independent of endogenous substrates, with the exception of exchange with atmospheric air. In this way major drawbacks of calculation and interpretation of results from measurements of DNA synthesis by isotope tracer techniques... 

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