Interactions of sulphide and other ligands with cytochrome c oxidase. An electron-paramagnetic-resonance study.

The effect of sulphide on resting oxidized cytochrome c oxidase was studied by both e.p.r. and optical-absorption spectroscopy. Excess sulphide causes some reduction of cytochrome a, CuA and CuB, and the formation of the cytochrome a3-SH complex after about 1 min. After several hours in the presence of excess sulphide only the e.p.r. signals due to low-spin ferricytochrome a3-SH persist, giving a partially reduced species. Re-oxidation of this partially reduced sulphide-bound enzyme by ferricyanide makes all of the metal centres except CuB detectable by e.p.r. We conclude that sulphide has reduced and binds to CuB as well as to ferricytochrome a3. Sulphide binding to cuprous CuB may raise its mid-point potential and make re-oxidation difficult. Addition of reductant (ascorbate + NNN'N'-tetramethyl-p-phenylenediamine) and sulphide together to the oxidized resting enzyme produces a species in which cytochrome a and CuA are nearly completely reduced and cytochrome a3 is e.p.r.-detectable as approx. 80% of one haem in the low-spin sulphide-bound complex. The g = 12 signal of this partially reduced derivative is almost unchanged in magnitude relative to that of the resting enzyme; this suggests that the g = 12 signal may arise from less than 20% of the enzyme and that it may be relatively unreactive to both ligation and reduction. Such a reactivity pattern of the g = 12 form of the oxidase is also demonstrated with the ligands F- and NO, which are thought to bind to cytochrome a3 and CuB respectively.     

Magnetic-circular-dichroism studies of haem a and its derivatives.

1. The Thy-1 membrane glycoproteins from rat thymus and brain bound deoxycholate to 24% of their own weight as measured by equilibrium dialysis. The binding occurred co-operatively at the critical micelle concentration of deoxycholate, suggesting that the glycoproteins bind to a micelle, and not to the detergent monomer. 2. From sedimentation-equilibrium and deoxycholate-binding data the molecular weights of the glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoprotein monomers were calculated to be 18700 and 17500 for thymus and brain Thy-1 glycoproteins respectively. The molecular weight of the polypeptide part of the glycoprotein is thus 12500. 3. In the absence of deoxycholate, brain or thymus Thy-1 glycoprotein formed large homogeneous complexes of mol. wt. 270000 or 300000 respectively. The sedimentation coefficient of these was 12.8 S. The complex was only partially dissociated by 4M-guanidinium chloride. 4. After cleavage of brain or thymus Thy-1 glycoprotein with CNBr, two peptides were clearly identified. They were linked by disulphide bonds and both contained carbohydrate. This cleavage suggests there is only one methionine residue per molecule, which is consistent with the above molecular weights and the known amino acid composition.     

A comparison of lipoprotein lipase activity and adipocyte differentiation in growing male rats.

During adipose tissue development changes in lipoprotein lipase activity per adipocyte precede significant changes in fat cell size. Lipoprotein lipase activity per adipocyte increases fourfold from the second to seventh postnatal week. Furthermore, when isolated adipocytes and stromal--vascular cells are prepared by collagenase digestion of adipose tissue, there is a progressive shift in enzyme activity during development from the stromal-vascular compartment to the adipocyte fraction. The data support the concept that during normal development a "bed" of preadipocytes is synthesized during the suckling period. The data further suggest a regulatory role for lipoprotein lipase in the control of "lipid-filling" during early postnatal development.     

The evolution of conspicuous coloration: Some experiments in bad taste

Unpalatable species are often brightly coloured. Such aposematic coloration may have evolved because predators can learn to avoid conspicuous prey more readily than cryptic prey. Experiments on young male chicks are described and the results are consistent with this hypothesis.     

Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy.

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)--CN in the partially reduced state. Hence as haem a3+(3)--CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)--CN . Cu+a3.     

The electron-transfer reaction between azurin and the cytochrome c oxidase from Pseudomonas aeruginosa.

A stopped-flow investigation of the electron-transfer reaction between oxidized azurin and reduced Pseudomonas aeruginosa cytochrome c-551 oxidase and between reduced azurin and oxidized Ps. aeruginosa cytochrome c-551 oxidase was performed. Electrons leave and enter the oxidase molecule via its haem c component, with the oxidation and reduction of the haem d1 occurring by internal electron transfer. The reaction mechanism in both directions is complex. In the direction of oxidase oxidation, two phases assigned on the basis of difference spectra to haem c proceed with rate constants of 3.2 X 10(5)M-1-S-1 and 2.0 X 10(4)M-1-S-1, whereas the haem d1 oxidation occurs at 0.35 +/- 0.1S-1. Addition of CO to the reduced enzyme profoundly modifies the rate of haem c oxidation, with the faster process tending towards a rate limit of 200S-1. Reduction of the oxidase was similarly complex, with a fast haem c phase tending to a rate limit of 120S-1, and a slower phase with a second-order rate of 1.5 X 10(4)M-1-S-1; the internal transfer rate in this direction was o.25 +/- 0.1S-1. These results have been applied to a kinetic model originally developed from temperature-jump studies.     

The mitochondrial complex in cryptophyceae

The unitary nature of the mitochondrion and the characteristic flattened finger-like morphology of the cristae were demonstrated in the Cryptophyceae. Hemiselmis rufescens contained an unbranched vermi-form mitochondrion in contrast to the variously branched complex, comprising an interconnected peripheral and central reticulum, in Chroomonas sp. and strains of Cryptomonas. The systematic value of the shape and distribution of the mitochondria in the examined genera was suggested.     

The influence of snow conditions on the date of breeding of wading birds in north-east Greenland

The young of wading birds that breed in some parts of the arctic hatch when their insect food supply is most abundant. This appears to be the ultimate factor determining the date of egg-laying. In various other arctic birds the date of breeding may be influenced, directly or indirectly, by the time at which snow or ice melts. The Joint Biological Expedition to N.E. Greenland 1974 studied Ringed plovers Charadrius hiaticula , Dunlins Calidris aplina , Sanderlings Calidris alba , Knots Calidris canutus , and Turnstones Arenaria interpres breeding in various areas within 80 km of each other.
In 1974 the thaw was two or three weeks later than normal in the region. The time at which snow cleared and the dates of breeding of the waders differed considerably between valleys: breeding was delayed in valleys that cleared late. In all areas egg-laying ceased early in July, probably because chicks hatching from eggs laid later would not be ready to migrate by the end of the summer. Hence the mean date and length of the egg-laying period were determined by the date of snow clearance from the breeding areas.
Studies of the potential food resources indicate that the waders in N.E. Greenland do not appear to time their breeding so that the young hatch at some peak of food abundance. Sufficient food resources are probably available over a long period of time.     

Acne and anticonvulsants.

The severity of acne and rate of excretion of sebum were assessed in 243 patients with epilepsy taking various anticonvulsants who were in hospital long term and in matched controls derived from a normal population of 2176 people. Neither the prevalence of acne nor the sebum excretion rate significantly increased in the patients compared with the controls or in patients taking phenytoin compared with those not. It is concluded that anticonvulsant treatment does not cause acne.