Some hormonal effects on myocardial phosphate efflux.

The effects of 23 agonists on the rates of cellular 32P efflux and lactate dehydrogenase (LDH) release were tested in a perfused rat heart preparation which had been prelabelled in vitro with [32P]Pi. Some 13 compounds produced detectable changes at high doses within 10 min, and in most cases a polyphasic response was observed. Six classes of compound gave rise to substantial effects, as follows. Catecholamines and glucagon produced a transient initial stimulation of Pi efflux, followed by a long-term inhibition of Pi transport and an increased rate of LDH release. These effects were clearly different from the response seen after treatment with dibutyryl cyclic AMP, which had a slower, stimulatory, effect on Pi output in doses which gave rise to a pronounced inotropic effect, and produced a marked increase in both coronary flow and LDH release. Carbachol also gave rise to a large transient stimulation of Pi efflux, which was followed by smaller sustained increase in Pi output without any obvious effect on LDH release. Dibutyryl cyclic GMP had no effect on Pi efflux or LDH release. Insulin decreased the rate of Pi efflux, although the loss rate partially recovered towards the control value after prolonged exposure to the hormone. Insulin had no obvious inotropic effects and produced no change in the rate of LDH release. Corticosteroids increased the rate of Pi efflux, although the loss rate partially declined towards the control value with prolonged exposure to the hormones. Corticosteroids produced a very slight inotropic response, and large doses sometimes increased the rate of LDH release from the tissue. Aldosterone slightly stimulated Pi output. A small, transient and somewhat variable stimulation of Pi efflux was observed with vasopressin and angiotensin, whereas tri-iodothyronine was slightly inhibitory, but adenosine, histamine, spermidine, des-Asp1-angiotensin, prolactin, parathyroid substances, calcitonin and somatostatin had no significant effects under our experimental conditions. Ouabain stimulated Pi efflux in doses that had no detectable inotropic effect. It is suggested that Pi efflux involves the electroneutral transport of NaH2PO4 across the cardiac plasmalemma and that many of the hormonal effects might be explained by changes in the intracellular [Na+] and pH in addition to changes in the intracellular [Pi].

A common source of error in pH measurements.

Glass-electrode assemblies in which the reference half-cell contains a porous ceramic type of liquid junction are likely to produce misleading pH measurements under normal service conditions. The error arises from substantial liquid-junction potentials, associated with the porous ceramic plug, which vary with the ionic composition of the solution under test. The error is not revealed by conventional two-point calibration procedures, since the majority of standard buffer solutions have a similar total ionic strength, but will nevertheless be present when the unknown solution differs in ionic strength from the standardizing buffers. The size of the error is proportional to the ratio between the salt concentration in the standard buffers and the concentration present in the unknown solution, and varies from one electrode specimen to another. The fault was present in 24 out of 30 electrodes in normal use selected at random from seven laboratories, and the mean error was 0.2pH unit per 10-fold salt-concentration difference between standard and test solutions. It is estimated that errors of this order must be widespread in the recent literature. Older pH determinations are likely to be more reliable, since the original reference electrode design with a free-flowing liquid junction is apparently free from the artefact.

Aerobic lactate synthesis by cardiac muscle.

Cardiac lactate production under aerobic conditions is absolutely dependent upon the availability of extracellular pyruvate. In the steady state, aerobic lactate output is largely independent of cardiac work load, but increases slightly when octanoate is included in addition to pyruvate in the perfusion fluid. Transient episodes of supra-normal lactate production are seen after sudden increases in cardiac work output, and also after transitions from octanoate to pyruvate in the perfusion media. These pulses of lactate production are invariably associated with the slow activation of pyruvate dehydrogenase in response to a sudden change in cardiac metabolic state, and they are abolished by pre-perfusion with dichloracetate, which converts pyruvate dehydrogenase into the fully active form. A second, additional component of the lactate pulses is sensitive to pre-perfusion with the transaminase inhibitor aminooxyacetate. The size of the second component is markedly dependent upon the precise protocol adopted for the experiment, and these variations suggest that the second component is associated with a major redistribution of cardiac Krebs' cycle intermediates and amino acids following the initial exposure to pyruvate-containing media. Steadystate aerobic lactate production is insensitive to both dichloroacetate and aminooxyacetate, and is thought to result from a direct exchange of malate for oxaloacetate across the heart mitochondrial membranes.

Regulation of myocardial energy metabolism.

Closed aorta working hearts perfused with 1 mM pyruvate were subjected to a 4-fold increase in work load by raising the left atrial filling pressure. Citric acid cycle flux, pyruvate uptake, and oxygen consumption rose 3-fold when cardiac output was increased. In the first 40 sec after the transition tissue glutamate and citrate fell by 22 and 45%, respectively, and there were reciprocal decreases in malate and aspartate. The ratio of creatine phosphate/creatine declined by 50% within 30 sec, with a corresponding increase in inorganic phosphate, but the fall in the ATP/ADP ratio was only 10%. During the first 10 sec the surface fluorescence from cardiac pyridine nucleotides fell by 30% and this change was synchronous with a sharp decline in the calculated adenine nucleotide phosphate potential. This suggests that heart mitochondrial respiration is controlled by the cytosolic phosphate potential, and that a state 4 to state 3 transition occurs when cardiac output is increased. Apparent disequilbrium of creatine phosphokinase can be explained by the compartmentation of most of the cardiac ADP within the mitochondria. Citric acid cycle flux was coordinated by activational interactions at citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, but a transient imbalance between the individual cycle steps leads to a sharp peak of lactate production shortly after the work transition.

Simulation of biogenic amine metabolism in the brain.

The metabolism of a number of biogenic amines has been simulated by using data obtained from studies of the individual enzymes from pig brain. It is shown that beta-hydroxylated amines such as noradrenaline and octopamine are metabolized primarily to the alcoholic metabolite whereas amines lacking this group [e.g. dopamine (3,4-dihydroxyphenethylamine) and 5-hydroxytryptamine] are metabolized at low concentrations to give the corresponding acid. Increase in the amine concentration results in an increase in the proportion of the alcoholic metabolite formed and this may in part account for the effects of the drug reserpine on amine metabolism. The effects of disulfiram (Antabuse) and ethanol (acting through its metabolite acetaldehyde) on amine metabolism may be understood in terms of this simulated model. It is shown that drugs that affect this system also cause alterations in the steady-state concentrations of the intermediate aldehydes and the possible implications of this are discussed.

Purification of yeast isocitrate dehydrogenase.

The NAD-linked isocitrate dehydrogenase from baker's yeast was purified to homogeneity (as judged by gel filtration and polyacrylamide-gel electrophoresis) with an overall yield of 50% by using dilute solutions of the allosteric effector (AMP) to elute the enzyme specifically from CM-cellulose. This method preserves the allosteric properties of the crude enzyme. Although the pure enzyme shows only a single band on electrophoresis in the presence of sodium dodecyl sulphate, two types of subunit are observed in 8m-urea. The isoelectric point of the enzyme rises during purification, and this may reflect the partial loss of an additional low-molecular-weight component. Values are included for the amino acid composition and extinction coefficients of the pure enzyme.

Anomalous behavior of yeast isocitrate dehydrogenase during isoelectric focusing.

Isoelectric focusing of yeast isocitrate dehydrogenase apparently reveals a number of ;isoenzymes'. These have isoelectric points near pH5.5 in crude material, but during purification the mean isoelectric point progressively rises to pH7.0 and the band pattern changes. The shift in isoelectric point during purification is apparently genuine, since it is also manifested in the electrophoretic and chromatographic properties of the enzyme. The multiple forms, however, are an artifact, generated by exposure of the enzyme to Ampholine, since their activities vary with the protein/Ampholine ratio and they cannot be observed in any system from which Ampholine is excluded. There are no detectable isoenzymes of yeast isocitrate dehydrogenase.

Purification and properties of the nicotinamide-adenine dinucleotide phosphate-dependent isocitrate dehydrogenase from pig liver cytoplasm.

The NADP-dependent isocitrate dehydrogenase from pig liver soluble fraction was purified over 500-fold with an overall yield of 25%. The purified enzyme, which is homogeneous by all the usual criteria, has a molecular weight of about 75000 and is composed of two identical subunits. This has been demonstrated by ultracentrifugation, fluorescence titration and peptide ;fingerprinting'. The maximal turnover number, extinction coefficients at 280nm and 260nm and amino acid analysis are described.

Reduction of oxaloacetate by pig liver isocitrate dehydrogenase.

Pure isocitrate dehydrogenase from pig liver cytoplasm catalyses the reduction of oxaloacetate by NADPH at a rate comparable with that observed for the usual substrates. The products are NADP and D-malate, the 'unatural' isomer. High concentrations of magnesium (25 mM) are necessary for maximal activity, and the reaction is not appreciably reversible. These results are discussed in connection with the inhibition of the enzyme by mixtures of glyoxylate and oxaloacetate. The reduction is not thought to be of physiological importance.

An automatic apparatus for the study of enzyme kinetics.

A continuous-flow apparatus is described for automatically plotting substrate saturation curves, and is suitable for use with a variety of enzymes. A linear concentration gradient of the variable substrate is combined with a fixed proportion of the other substrates and the enzyme, and after passing through a reaction coil the product concentrations are measured spectrophotometrically. Use of a 4cm. flow cell and modified spectrophotometer permits accurate measurement of NADH concentration in the region of 0.1mum. Precise control over reaction times and substrate concentration is achieved by using power-driven syringes with an integral mixer. Specimen results are given for yeast alcohol dehydrogenase.