Regulation of the pyruvate dehydrogenase complex by Ca2+ within toluene-permeabilized heart mitochondria.

(1) Rat heart mitochondria, permeabilized to all low Mr solutes by toluene treatment, have been used to study the regulation in situ of the phosphatase and kinase components of the pyruvate dehydrogenase complex (PDH) by Ca2+. (2) Inactivation of the complex, resulting from phosphorylation by the kinase, and reactivation induced by the phosphatase, were both apparent first-order processes. This behaviour of the phosphatase differs from that observed with toluene-permeabilized adipose tissue mitochondria (Midgley, P.J.W., Rutter, G.A. and Denton, R.M. (1987) Biochem. J. 241, 271-377) where a 'lag phase' preceded reactivation of inactive complex. Further, reactivation due to phosphatase activity was stimulated by Ca2+ only at subsaturating Mg2+ concentrations, in contrast with the extracted enzyme which is stimulated by Ca2+ at all Mg2+ concentrations. (3) Maximum values of half-times observed for inactivation and reactivation were about 10 and 15 s, respectively, at 30 degrees C. (4) At Mg2+ concentrations where effects of Ca2+ on the activity of the phosphatase were apparent, no effect of Ca2+ on the activity of the kinase could be detected. (5) The sensitivity of the phosphatase to [Ca2+] was essentially unchanged in the presence of either ADP or ATP, with half-maximal effects at 0.7 microM in each case.

Effects of Ca2+ on the activities of the calcium-sensitive dehydrogenases within the mitochondria of mammalian tissues.

Three important dehydrogenases in the mitochondria of mammalian tissues are activated by Ca2+ ions: these are pyruvate dehydrogenase, NAD-isocitrate dehydrogenase, and oxoglutarate dehydrogenase. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+ in rat hearts and livers that this results in a parallel rise in the intramitochondrial concentration of Ca2+. In this way, pyruvate oxidation and citric acid cycle flux are stimulated and there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP.

Effects of Ca2+ and Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within toluene-permeabilized mitochondria.

Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.

Hormonal regulation of fluxes through pyruvate dehydrogenase and the citric acid cycle in mammalian tissues.

Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. Activation can also be demonstrated within permeabilized and intact mitochondria. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+. In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP. In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism.

Exofacial photoaffinity labelling of the human erythrocyte sugar transporter.

The 4-azidosalicylate derivative of 1,3-bis(D-mannos-4'-yloxy)-2-[2-3H]propylamine (ASA-[2-3H]BMPA) has been tested as a photoaffinity label for the sugar transporter in human erythrocytes. When photolysed in the presence of intact erythrocytes, ASA-[2-3H]BMPA covalently binds to the exofacial surface of the transporter. This labelled protein appears as a broad band in the 4.5 region in sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The peak of radiolabel incorporation gives an apparent Mr of approx. 50 000 on 5-20% acrylamide gels. The binding is 80% inhibitable by 320 mM 4,6-O-ethylidene-D-glucose, by 320 mM D-glucose and by 50 microM cytochalasin B. Photoirradiation of a saturating concentration of ASA-BMPA in the presence of erythrocytes results in a 25-30% loss of D-galactose transport activity. From transport inactivation data and estimations of the amount of ASA-[2-3H]BMPA binding to the transporter it is calculated that there are approx. 220 000 exofacial hexose-transport binding sites per erythrocyte. The labelling of the transporter has been carried out using freshly drawn blood and 4-weeks-old transfusion blood. No change in the binding profile on SDS-polyacrylamide gel electrophoresis was observed. Proteolytic digestion of the ASA-[2-3H]BMPA-labelled transporter with either trypsin or alpha-chymotrypsin results in the appearance of a labelled 19 kDa fragment on SDS-polyacrylamide gel electrophoresis.

Interaction of hydrophobic bis (D-mannose) derivatives with adipocyte and erythrocyte sugar transport systems.

The inhibition of sugar uptake by a series of hydrophobic bis(D-mannose) derivatives has been measured in rat adipocytes. When the D-mannose moieties of the bis compounds are separated by a hexane bridge the transport inhibition constant (Ki) is greater than for a decane-bridged molecule. This is probably due to the increased hydrophobicity of the bridge of the decane-bridged compound. The enhancement in affinity due to the second sugar in the bis(D-mannose) derivatives is probably only 2-fold, since half reduction of the bis(D-mannosyloxy)hexane increases Ki approx. 2-3-fold. N'-DNP-1,3-bis(D-mannos-4'-yloxy)propyl-2-amine has very high affinity in insulin-treated cells. The affinity is approx. 1000-fold higher than for D-mannose. This enhancement is probably due to the hydrophobicity of the DNP group. The distance from the sugar to the hydrophobic group is important because an increase in Ki occurs if an aminocaproyl spacer is introduced between the DNP group and 1,3-bis(D-mannos-4'-yloxy)propyl-2-amine. Aminocaproyl and glycyl spacers also increase the Ki for NAP derivatives of 1,3-bis(D-mannos-4'-yloxy)propyl-2-amine. Each of the hydrophobic bis(D-mannose) derivatives has a lower Ki in insulin-treated cells. This may be due to an insulin responsive hydrophobic interaction between the hydrophobic portion of the sugar and a hydrophobic domain in the transport system. The inhibition constants for the hydrophobic bis(D-mannose) compounds have also been measured in human erythrocytes.

A new class of sugar analogues for use in the investigation of sugar transport.

D-Mannose derivatives have been synthesised which are crosslinked through their C-4 hydroxyls to propyl-2-amine. Coupling to the amino group gave a fluorodinitrobenzene derivative, a nitroazidophenyl derivative and an azidosalicylamide derivative. Each of these derivatives was shown to have high affinity for the human erythrocyte sugar transport system. The affinity constant for the nitroazidophenyl derivative was not altered by temperature changes. In rat adipocytes treated with insulin, the affinity constants for the derivatives were up to 1000-fold lower than for the parent sugar. In the absence of insulin the affinity constants for the derivatives, but not for D-mannose, were 3-times higher than in insulin-treated cells. By preparation of radiolabelled derivatives we have shown that the compounds are not transported either by erythrocytes or by adipocytes. Thus the crosslinked sugars are good outside-specific analogues.

Light-scattering from detergent-complexed biological macromolecules.

A theory is described for Rayleigh light-scattering from solutions of detergent-complexed macromolecules applicable to measurements carried out under conditions of Donnan equilibrium. The theory shows that when scattering measurements are made on detergent-solubilized macromolecules in the presence of detergent micelles the apparent Mr is dependent on the extent of detergent binding and effective charge on the detergent-macromolecule complex and the micellar charge and aggregation number. Equations are given for the apparent Mr of the macromolecule under limiting conditions of high salt and low salt concentration. Low-angle laser-light-scattering measurements were made on lysozyme complexed with sodium n-dodecyl sulphate both in the absence and in the presence of detergent micelles. These experimentally obtained data were used in conjunction with the detergent-binding isotherm to test the theory at high ionic strength. Light-scattering measurements were also made on detergent-saturated complexes as a function of ionic strength and pH. The results are in reasonable accord with both the qualitative and the quantitative predictions of the theory.