Biochimica et biophysica acta最新文献

The influence of the external Na⁺ and K⁺ concentration and of ouabain on I⁻ uptake and release was studied in cow-thyroid slices blocked with propylthiouracil. It could be shown that the I⁻ transport is dependent on the presence of both Na⁺ and K⁺ ions. However, the dependence on Na⁺ seems to be more direct: the effect of removal of K⁺ and of the application of ouabain set in only after a latency period while the effect of Na⁺ removal was immediate. The half-time of I⁻ release due to removal of Na⁺ from the medium was markedly shorter than that due to K⁺ removal or that of the release caused by ouabain. However, the half-times in the latter two cases were similar. A thyroid slice kept for a long time under ouabain poisoning in a solution wherein 90% of the Na⁺ was replaced by Li⁺ was still able to accumulate I⁻ for a short time when brought back into a medium of normal Na⁺ content in spite of the uninterrupted presence of ouabain. The possible connections between the K⁺-Na⁺ transport mechanism and the I⁻ transport are discussed.

In principle two basically different relations can be thought of. Either the I⁻ transport is directly coupled with the Na⁺K⁺ exchange mechanism or the cation-concentration differences created by the Na⁺K⁺ transport system are a prerequisite for the I⁻ transport. The present results are taken to favour the latter assumption.

• 1.

  1. An enzyme which catalyses the transamination between γ-hydroxyglutamate and α-ketoglutarate was extracted from rat-liver acetone powder and purified in excess of 15-fold. Fractionation of the enzyme from a separate aspartate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) remained incomplete.

• 2.

  2. [2-¹⁴C]Pyruvate was incorporated into γ-hydroxy-α-ketoglutarate and γ-hydroxyglutamate in the presence of glyoxylate in rat-liver homogenate. Specific activity values of these three compounds indicated the occurence of the following pathway: Pyruvate + glyoxylate ⇌ γ-hydroxy-α-ketoglutarate ⇌ γ-hydroxyglutamate.

• 3.

  3. A further possible pathway of γ-hydroxy-α-ketoglutarate metabolism leading to malate and of γ-hydroxyglutamate to its decarboxylation product or to L-hydroxyproline were inferred and discussed.

• 1.

  1. This paper is concerned with the study of the formation of non-phosphorylated intermediates in digitonin particles prepared from rat-liver mitochondria.

• 2.

  2. Digitonin particles were preincubated with either β-hydroxybutyrate, sucinate or ferrocytochrome c for a short time at 20°. After cooling the preincubated mixture was incubated at 1° with ³²P_i and ADP. Under these conditions, the time course of [³²P]ATP synthesis is represented by a biphasic curve. The initial portion corresponds to a high rate of [³²P]ATP synthesis whereas the second portion corresponds to a slower rate.

• 3.

  3. The rapid initial [³²P]ATP synthesis is maximal for pH values ranging from 6.5 to 7.3. It is increased by compounds which increase the efficiency of oxidative phosphorylation in digitonin particles such as EDTA and serum albumin and is inhibited by inhibitors of oxidative phosphorylation such as 2,4-dinitrophenol or azide. It is delayed or considerably depressed when unlabeled P_i is added during the preincubation time prior to the addition of ³²P_i and ADP but is not affected by atractylate, a compound which is capable of specifically inhibiting the ATP-P_i exchange in digitonin particles without interfering with the ATP synthesis linked to respiration. Finally it is enhanced by low concentrations of oligomycin which at 1° have little effect on the rate and the yield of oxidative phosphorylation.

• 4.

  4. The effect of ³²P_i concentration on the initial rate of [³²P]ATP synthesis was determined: K_m = 1.7·10⁻³ M.

• 5.

  5. The experimental findings have been interpreted as follows: non-phosphorylated intermediates accumulate in digitonin particles during the oxidation of β-hydroxybutyrate, succinate or ferrocytochrome c. These intermediates are discharged upon addition of P_i and ADP, the net result of the reaction being the synthesis of ATP.