Robert W. Swick

Turnover of hepatic mitochondril ornithine aminotransferase and cytochrome oxidase using [14C] carbonate as tracer

Abstract The half-lives of the liver mitochondrial enzymes, ornithine aminotransferase (L-ornithine: 2-oxo acid aminotransferase, EC 2.6.1.13) and cytochrome oxidase (ferrocytochrome c : oxygen oxidoreductase, EC 1.9.3.1), have been determined under basal conditions by injecting rats with NaH 1 4 CO 3 and following the decay in specific radioactivity of arginine isolated from the purified enzymes. This technique gave half-lives for the two enzymes of 1.9 and 5.7 days, respectively. An identical half-life was obtained for cytochrome oxidase using δ-amino [ 3 H]levulinate as a tracer, thus indicating that the heme and apoprotein moieties of this enzyme turn over at the same rate. The degradation rate constant of ornithine aminotransferase under induced, steady-state conditions, i.e. in rats fed a diet containing 80% of casein, was not significantly different from that under basal conditions; hence, the increased amount of enzyme present under these conditions must be due to an increased rate of synthesis. Fractionation of mitochondria with digitonin and Lubrol WX, and comparison with marker enzymes, indicated that ornithine aminotransferase is located within the mitochondrial matrix.

Isolation and characterization of mitochondrial alanine aminotransferase from porcine tissue

Mitochondrial alanine aminotransferase L-alanine:2-oxoglutarate aminotransferase, EC 2.6.1.2) has been isolated in homogeneous form from both porcine liver and kidney cortex, but in low yield. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate or 8 M urea gave a single band. An isoelectric point of 8.5 +/- 0.5 and a molecular weight of 75--80 000 were obtained. The enzyme is specific for L-alanine and is inhibited by D-alanine, aminooxyacetate and cyclosterine. The Km for pyruvate and glutamate is 0.4 mM and 32 mM, respectively. These values are similar to those determined for the cytoplasmic enzyme; however, at high concentrations, both compounds strongly inhibit the mitochondrial enzyme, an inhibition not observed with cytosolic alanine aminotransferase. These characteristics and the fact that the mitochondrial alanine aminotransferase was inactivated by procedures effective in the preparation of the cytosolic enzyme, clearly differentiate the two proteins and further support different roles for the two alanine aminotransferases in vivo.

Unmasking of GDP binding sites on hamster brown adipose tissue mitochondria and uncoupling protein.

1. A rapid unmasking of GDP binding sites on brown adipose tissue (BAT) mitochondria was observed when hamsters acclimatized to 28 degrees C were exposed to a temperature of 4 degrees C for 2 hr. 2. No rapid unmasking of GDP binding sites was observed when hamsters housed at 22 degrees C were briefly exposed to 4 degrees C. 3. The amount of GDP bound to BAT mitochondria from hamsters increased during 2 weeks of exposure to 4 degrees C, but did not change between 2 weeks and 30 days of cold exposure. 4. Incubation of mitochondria with 10 mM Mg2+ prior to the GDP binding assay increased the subsequent GDP binding to BAT mitochondria from hamsters housed at 28, 22 or 4 degrees C, albeit to different degrees. 5. The amount of GDP bound to uncoupling proteins isolated from untreated and Mg(2+)-treated mitochondria of hamsters and rats was measured. Scatchard analyses of the binding of GDP to purified uncoupling protein indicate that increases in the number of binding sites due to Mg2+ treatment of mitochondria do not change the affinity of the protein for GDP.

Growth and protein turnover in animals

Protein turnover is a dynamic process involving most of the tissue proteins: the quantity of protein turned over each day is about 5 to 10 times the daily dietary requirement. Growth, i.e., the accumulation of protein, is the difference between protein synthesized and protein broken down and usually represents only a small fraction of the total protein turned over. Growth rates could be enhanced if protein synthesis, which is energetically expensive, could be increased or if protein degradation, which is energetically much less expensive, could be decreased. Small changes which would be compatible with good health would have significant economic effects on animal and meat production. Millward et al.10 showed that in well‐fed rats, there is a developmental fall in rates of skeletal muscle protein synthesis accompanied by a smaller but parallel decrease in the fractional degradation rate. The result of these changes is a decrease in growth rate with increasingage. Maruyama et al.9 using well‐fed broiler chi...

Brown adipose tissue from fetal rhesus monkey (Macaca mulatta): morphological and biochemical aspects

Brown adipose tissue (BAT) from fetal rhesus monkeys microscopically resembled adult rodent BAT containing multiocular fat cells with numerous mitochondria. Mitochondrial carnitine palmitoyl transferase activity was lower than that in adult rodents and adenine nucleotide translocase activity was similar to that reported for rats. Rhesus monkey BAT mitochondria (BATM) possess an uncoupling protein that is characteristic of BAT as evidenced by the binding of [3H]GDP, the inhibition by GDP of the high Cl- permeability or rapid alpha-glycerol-3-phosphate oxidation. Electrophoretic analysis of BATM showed the presence of a 32,000 mol.wt protein which was enriched by procedures established for the isolation of BATM uncoupling protein.

Characteristics of growth and protein synthesis in skeletal muscles of normal and dystrophic chickens

Several characteristics of cellular growth and protein synthesis were measured in vivo in the breast and leg muscles of normal (line 454) and hereditary muscular dystrophic (line 455) New Hampshire chickens at different ages. Fractional rates of protein synthesis (ks) were calculated from the rate of incorporation of labeled tyrosine administered continuously via a subcutaneous injection of an emulsion of L-[U-14C]tyrosine in sesame oil. Only the breast muscle showed signs of gross hypertrophy. The hypertrophy involved an abnormally rapid accumulation of protein, DNA and RNA. In dystrophic chickens, ks was at least 170% of the normal rates at 2, 5 and 52 weeks. The elevated ks was presumably a reflection of the higher DNA and RNA concentrations relative to those in normal birds. Because the RNA/DNA ratio and the activities of RNA and DNA were similar to values observed in normal birds, there may be no defect in the synthesis machinery per se.

Determination of the Average Degradation Rate of Mixtures of Protein

Rates of breakdown of proteins can be determined from the decay of label in protein after injection of a non-reutilisable precursor. With homogeneous proteins the decay is exponential which, plotted on semi-log graph paper, gives a straight line whose gradient is the fractional rate of breakdown. With mixtures of protein, such as occur in whole tissues, the semi-log plot of radioactivity against time is curved. The apparent rate of breakdown, therefore, varies with the duration of the experiment, becoming slower as the period of measurement becomes longer (1–4). We have attempted to obtain a unique value for the average rate of turnover of rat liver protein from the decay of label in protein after 14CO 3 = injection.