The chemistry of flavines and flavoproteins. Photoreduction of flavines by amino acids

1. Flavines are photoreduced through their triplet states by amines and amino acids (e.g. EDTA and dl-phenylglycine). The anaerobic photoreduction of FMN and several other flavines with dl-phenylglycine was analysed in terms of a detailed kinetic scheme. 2. The reaction produces equimolar amounts of benzaldehyde, carbon dioxide and reduced flavine. 3. The sensitivity of the rates to substituents in the dl-phenylglycine can be described by a Hammett rho-value of -1.1. 4. Phenylacetic acid behaves differently from dl-phenylglycine or benzylamine towards a series of flavines. 5. The photoreductions are quenched by several aromatic compounds. From the effects of light-intensity and temperature, and by comparison with potassium iodide quenching, it is concluded that inhibition by the aromatic compounds is not simply a collisional process. 6. FAD reacts more slowly than FMN both in the photoreduction and in dark reduction by NADH. Urea and dimethyl sulphoxide decrease the intramolecular interaction in FAD, but they have no effect on the rate of dark reduction of FAD compared with FMN. In contrast, the photoreduction of FAD is quicker in urea.     

Nucleic acid metabolism in rat hematopoietic tissues during and after prolonged administration of different doses of tritium oxide

During the long-term administration to rats of tritium oxide in doses of 0.37, 0.925 and 1.85 MBq/g body mass the content of karyocytes and nucleic acids in the bone marrow and spleen was decreased, the rate of their biosynthesis changed, the DNA structure impaired, the content of salt-soluble polydeoxynucleotides increased, and DNAases activated. The observed changes were function of dose. After the end of the administration of the isotope the animals which had received a lesser tritium dose exhibited a more rapid and complete recovery.     

Motor neuron degeneration in a mouse model of seipinopathy

Heterozygosity for missense mutations (N88S/S90L) in BSCL2 (Berardinelli–Seip congenital lipodystrophy type 2)/Seipin is associated with a broad spectrum of motoneuron diseases. To understand the underlying mechanisms how the mutations lead to motor neuropathy, we generated transgenic mice with neuron-specific expression of wild-type (tgWT) or N88S/S90L mutant (tgMT) human Seipin. Transgenes led to the broad expression of WT or mutant Seipin in the brain and spinal cord. TgMT, but not tgWT, mice exhibited late-onset altered locomotor activities and gait abnormalities that recapitulate symptoms of seipinopathy patients. We found loss of alpha motor neurons in tgMT spinal cord. Mild endoreticular stress was present in both tgMT and tgWT neurons; however, only tgMT mice exhibited protein aggregates and disrupted Golgi apparatus. Furthermore, autophagosomes were significantly increased, along with elevated light chain 3 (LC3)-II level in tgMT spinal cord, consistent with the activation of autophagy pathway in response to mutant Seipin expression and protein aggregation. These results suggest that induction of autophagy pathway is involved in the cellular response to mutant Seipin in seipinopathy and that motoneuron loss is a key pathogenic process underlying the development of locomotor abnormalities.     

Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.     

Spin label and lanthanide binding sites on glyceraldehyde-3-phosphate dehydrogenase.

The electron spin resonance spectrum of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase spin-labelled with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl has two components. One component is due to a spin label highly immobilized on the enzyme surface and the other to a nitroxyl group able to tumble more rapidly. The spin-labelled enzyme is inactive. Selective modification of the active site cysteine residue (149) and determinations of total sulphydryl content implicate this residue as the site of the immobile spin-label. The mobile spin label is attached to another sulphydryl group. Crystallographic studies on the human muscle enzyme (Watson, H.C., Duee, E. and Mercer, W.D. (1972) Nat. New Biol., 240, 130) have located a binding site for samarium ion in the active centre. Addition of the paramagnetic gadolinium ion to spin-labelled enzyme reduces the intensity of both the spin label signals (by 72% for the mobile and by 11% for the immobile component). This indicates that the metal ion site (Kd equals 0.7 mM) is close to both types of spin label. Measurements of the effect of gadolinium-protein binding on the relaxation rate of solvent water protons enable the enzyme-bound spin label-metal ion distances to be tentatively estimated as 15 angstrom.     

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain,and the ionophore R02-2985 (X537A) in the isolated,perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using ³¹P-NMR. Mechanical activity was increased by infusion of ouabain (0.9?7.0·10^?5 M), the ionophore R02-2985 (1·10^?5 M) or epinephrine (5·10^?8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ($\text{[}\text{ATP}\text{]}\text{[ADP][P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}$), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.     

Creatine kinase kinetics,ATP turnover,and cardiac performance in hearts depleted of creatine with the substrate analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% of the creatine substrate analogue β-guanidinopropionic acid for 6–10 weeks. ³¹P-NMR investigation of isolated, glucose-perfused working hearts showed a 90% reduction in [phosphocreatine] from 22.2 to 2.5 μmol/g dry wt in guanidinopropionic acid-fed animals but no change in [P_i], [ATP], or intracellular pH. The unidirectional exchange flux in the creatine kinase reaction (direction phosphocreatine → ATP) was measured by saturation transfer NMR in hearts working against a perfusion pressure of 70 cm of water. This exchange was 10 μmol/g dry wt per s in control hearts and decreased 4-fold to 2.5–2.8 μmol/g dry wt per s in hearts from guanidinopropionic acid-fed animals. Oxygen consumption and cardiac performance were measured in parallel experiments at two perfusion pressures, 70 and 140 cm. No significant differences were observed in oxygen uptake or in any of the performance criteria between hearts from control and guanidinopropionic acid-fed rats at either workload. Assuming an ADP:O ratio of 3, the oxygen consumption measurements correspond to ATP turnover rates of 4.2–7.8 μmol/g dry per s. These rates are 1.5–3-times greater than the rate of the phosphocreatine → ATP exchange in hearts from guanidinopropionic acid-fed rats. These data suggest that phosphocreatine cannot be an obligate intermediate of energy transduction in the heart.     

Increased lipolysis and energy expenditure in a mouse model with severely impaired glucagon secretion

Background

Secretion of insulin and glucagon is triggered by elevated intracellular calcium levels. Although the precise mechanism by which the calcium signal is coupled to insulin and glucagon granule exocytosis is unclear, synaptotagmin-7 has been shown to be a positive regulator of calcium-dependent insulin and glucagon secretion, and may function as a calcium sensor for insulin and glucagon granule exocytosis. Deletion of synaptotagmin-7 leads to impaired glucose-stimulated insulin secretion and nearly abolished Ca²⁺-dependent glucagon secretion in mice. Under non-stressed resting state, however, synaptotagmin-7 KO mice exhibit normal insulin level but severely reduced glucagon level.

Methodology/Principal Findings

We studied energy expenditure and metabolism in synaptotagmin-7 KO and control mice using indirect calorimetry and biochemical techniques. Synaptotagmin-7 KO mice had lower body weight and body fat content, and exhibited higher oxygen consumption and basal metabolic rate. Respiratory exchange ratio (RER) was lower in synaptotagmin-7 KO mice, suggesting an increased use of lipid in their energy production. Consistent with lower RER, gene expression profiles suggest enhanced lipolysis and increased capacity for fatty acid transport and oxidation in synaptotagmin-7 KO mice. Furthermore, expression of uncoupling protein 3 (UCP3) in skeletal muscle was approximately doubled in the KO mice compared with control mice.

Conclusions

These results show that the lean phenotype in synaptotagmin-7 KO mice was mostly attributed to increased lipolysis and energy expenditure, and suggest that reduced glucagon level may have broad influence on the overall metabolism in the mouse model.