Inhibition of exogenous NADH oxidation in plant mitochondria by chlorotetracycline in the presence of calcium ions

Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca²⁺. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca²⁺. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca²⁺. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn²⁺ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca²⁺ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca²⁺-chelators is not reversible or is enhanced by the addition of excess Ca²⁺. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca²⁺. It is suggested that a neutral 1:2 complex is formed between Ca²⁺ and chlorotetracycline which can substitute for Ca²⁺ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase.     

Interaction of fatty acids with lipid bilayers

We present a method by which it is possible to describe the binding of fatty acids to phospholipid bilayers. Binding constants for oleic acid and a number of fatty acids used as spectroscopic probes are deduced from electrophoresis measurements. There is a large shift in $\text{p}\text{K}$ value for the fatty acids on binding to the phospholipid bilayers, consistent with stronger binding of the uncharged form of the fatty acid. For dansylundecanoic acid, fluorescence titrations are consistent with the binding constants derived from the electrophoresis experiments. For 12-(9-anthroyloxy)stearic acid, fluorescence and electrophoresis data are inconsistent, and we attribute this to quenching of fluorescence at high molar ratios of 12-anthroylstearic acid to phospholipid in the bilayer.     

A homopolymer of the potent antitumor drug 2′-deoxy-5-fluorouridylate

Poly(5-fluoro-2′-deoxyuridylic acid) was synthesized and its properties were compared with those of poly(dT) and poly(dU). It readily complexed with poly(dA). The 1:1 complex melted at about 20°C lower than poly(dA) · poly(dT). A triple-stranded helix, poly(dA)·2 poly(dF⁵U) was formed only in high salt (2.0 M NaCl).     

Erythrocyte Lii-Nao countertransport system Inhibition by N-ethylmaleimide probes for a conformational change of the transport system

Human erythrocytes were treated by a series of SH-reagents, including maleimides, iodo compounds, mercurials and oxidizing agents. Rates of Li efflux into Na-rich medium, Li leak and Li_i-Na_o countertransport were then determined. Of the 13 different reagents studied, only N-ethylmaleimide, iodoacetamide and iodoacetate inhibited selectively the countertransport activity. The effect of the various reagents indicates that the sensitive SH-groups of the countertransport system are not externally exposed. N-Ethylmaleimide was used to probe for changes elicited by substrate cations in Li_i-Na_o countertransport. In Na- and Li-free medium, inhibition of Li_i-Na_o countertransport by N-ethylmaleimide of 35% was reached within 2 s. In Na or Li medium, maximal inhibition was twice as great, but was attained much more slowly, within 10 min. Kinetic data and Hill plot analysis indicate the involvement of two classes of SH-groups: one expressed in the various media with and without substrate cations, and an additional one, which becomes specifically available to N-ethylmaleimide in the presence of external Na or Li. The affinity of Na to the site promoting inhibition by N-ethylmaleimide (apparent K_m  12 mM) is higher than the affinity of Na to its external countertransport site (apparent K_m  25 mM), as reported by Sarakadi, B., Alifimoff, J.K., Gunn, R.B. and Tosteson, D.C. (1978) J. Gen. Physiol. 72, 249–265). Reactivity of $\text{N-}\text{ethyl}\text{[}{}^{14}\text{C}\text{]}\text{maleimide}$ was not modified by the media tested. It is concluded that external Na and Li cause a conformational change in the protein(s) of the countertransport system in human erythrocytes.     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Transbilayer distribution of phospholipids in photoreceptor membrane studied with trinitrobenzenesulfonate alone and in combination with phospholipase D

In a further study of the transbilayer distribution of phospholipids in rod disk membranes, the amino group reagent, trinitrobenzenesulfonate, and the phospholipid-hydrolyzing enzyme, phospholipase D, have been used alone and in combination.Under carefully defined conditions (1 mM trinitrobenzenesulfonate, pH 7.4, 20°C, darkness), trinitrobenzenesulfonate yields limited final levels of modification of phosphatidylethanolamine and phosphatidylserine, suggesting only minor reagent penetration and membrane disturbance under these conditions.Treatment of stacked disks with trinitrobenzenesulfonate under these conditions leads to a biphasic modification of the a aminophospholipids. Relatively fast (less than 1 h) modification of 50% phosphatidylethanolamine and 40% phosphatidylserine occurs, slowly rising (approx. 3 h) to 60 and 50%, respectively.Extensive treatment of stacked disks with phospholipase D leads to the hydrolysis of 55% phosphatidylcholine and 50% phosphatidylethanolamine, while phosphatidylserine is hardly attacked by this enzyme.Treatment of stacked disks with trinitrobenzenesulfonate after prior treatment with phospholipase D leads to no further modification than that maximally obtained with either reagent alone: about one-half of the three major phospholipid classes is accessible. Although both reagents differ greatly in molecular size, mode of action and other properties, they apparently see the same pool of phosphatidylethanolamine, their joint substrate. Considering that we start with the original right-side-out configuration, that all phospholipids can in principle be modified (no shielding) and that the membrane remains essentially intact, we conclude that the accessible lipid pool represents the outer face of the disk membranes.These results confirm our earlier conclusions from treatment with three phospholipases that the three major phospholipids are nearly symmetrically distributed over the two faces of the disk membrane.The divergence with the conclusions of other investigators is most likely explained by their use of disk membranes (disk vesicles) in which the original phospholipid distribution had not been maintained and/or of conditions under which trinitrobenzenesulfonate markedly penetrates the membrane.     

The nature of the changes in liver mitochondrial function induced by glucagon treatment of rats. The effects of intramitochondrial volume,aging and benzyl alcohol

(1) The effects of changes in the intramitochondrial volume, benzyl alcohol treatment and calcium-induced mitochondrial aging on the behaviour of liver mitochondria from control and glucagon-treated rats are reported. (2) The stimulatory effects of glucagon on mitochondrial respiration, pyruvate metabolism and citrulline synthesis could be mimicked by hypo-osmotic treatment of control mitochondria and reversed by calcium-induced aging of mitochondria or by treatment with 20 mM benzyl alcohol. Hypo-osmotic treatment increased the matrix volume whilst aging but not benzyl alcohol decreased this parameter. (3) Liver mitochondria from glucagon and adrenaline-treated rats were shown to be less susceptible to damage by exposure to calcium than control mitochondria and frequently showed slightly (15%) elevated intramitochondrial volumes. (4) Aging, benzyl alcohol and hypo-osmotic media increased the susceptibility of mitochondria to damage caused by exposure to calcium. (5) Glucagon-treated mitochondria were less leaky to adenine nucleotides than control mitochondria. (6) These results suggest that glucagon may exert its action on a wide variety of mitochondrial parameters through a change in the disposition of the inner mitochondrial membrane, possibly by stabilisation against endogenous phospholipase A₂ activity. This effect may be mimicked by an increase in the matrix volume or reversed by calcium-dependent mitochondrial aging.