No abstract

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20–30 % of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 × 106. Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 × 106, which contained a proportion of all the glycolytic enzyme studied, the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both‐the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6‐phosphate was reduced by the presence of unlabelled 3‐phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm, diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.

1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.

1.It was possible to find a steady-state lactate concentration in a closed-circuit heart perfusion containing added glucose. Insulin but not growth hormone (4 pglml) increased the equilibrium concentration in perfusate by 200 Ole. Intracellular lactate also remained constant and was about twice that in perfusate: this distribution is difficult to explain whether ionic or undissociated lactate is the permeating species.2. Pyruvate accumulated linearly in perfusate, reaching a peak after 30 min perfusion and then declined non-linearly. Insulin increased the peak concentration and growth hormone slowed the decline after the peak. The intracellular pyruvate concentration stayed constant and, except in the first few minutes, was lower than the perfusate concentration.3. Pyruvate addition a t zero time could inhibit pyruvate accumulation but altered neither the non-linear decline, the intracellular pyruvate level nor the lactate equilibrium. The [NAD]/[NADH]ratio, after 10 min of pre-perfusion on open circuit, was 23. During 2 h of perfusion the ratio rose slowly and then declined. If either insulin or growth hormone were present in the medium, the ratio fell to about 11 during the 6rst few minutes of perfusion and remained low for 90 min. These values could not easily be correlated with either the varying extracellular, or the constant intracellular [lactate]/[pyruvate] ratios.5. The glucose and oxygen consumption of the hearts during perfusion was measured, and the change in glycogen concentration in the tissue. It was then possible to draw up a balance sheet which indicated that the control hearts oxidied little other than carbohydrate for the 60 min of observation, but both insulin and growth hormone-treated hearts oxidised significant quantities of fatty acid during the first 15 min. It appears that growth hormone, like insulin, has an immediate effect on heart metabolism. The addition of pyruvate a t zero time appeared to stimulate carbohydrate oxidation in hearts perfused with insulin, even though the hormone had been present in the pre-perfusion fluid.6. Consideration of the variation in the extracellular [lactate]/[pyruvate] ratio with length of perfusion, and its lack of relationship to the intracellular [lactate]/[pyruvate] ratio, together with similar reports of other workers, leads us to conclude that neither lactate nor pyruvate establish a concentration gradient across the heart cell membrane which is based on passive diffusion. The discrepancy is particularly marked with respect to pyruvate. Our results strongly suggest that the extracellular [lactate]/[pyruvate] ratio cannot be used as an index of the cytoplasmic redox potential in perfused heart. 7.A tracer study of pyruvate metabolism in the reproducible non-steady-state conditions described can offer an explanation for the behaviour of the observed metabolic parameters : this study is reported in the following papers.The experiments which are reported in this and succeeding papers were designed to produce data that could be used to test a new metho...

The predicted presence in perfused rat hearts of a rapidly metabolized but hitherto unrecognized form of adenosine phosphate has been confirmed by specific radioactive labelling. The properties of the purified compound suggest that it is a heteropolymer of a small organic acid, phosphate and purine nucleoside in the proportions 1:4:1.