Kinetic studies on cytochrome c oxidase by combined epr and reflectance spectroscopy after rapid freezing.

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

Kinetic studies on cytochrome c oxidase by combined EPR and reflectance spectroscopy after rapid freezing

1. Techniques and experiments are described concerned with the millisecond kinetics of EPR-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O₂ or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy.2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (< 50 ms) approx. 0.5 electron equivalent per hame a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (> 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a.3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O₂ in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O₂ side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates.4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms, whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO.5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C. R., Hansen, R. E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477–2481). Both the low-spin (g = 3; 2.2; 1.5) and slowly appearing high-spin (g = 6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a₃, when it is present in a state of interaction with EPR-undetectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.     

ANO5 ensures trafficking of annexins in wounded myofibers

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.     

Nitric oxide and prostacyclin-dependent pathways involvement on in vitro induced hypothermia

Nitric oxide and prostacyclin are endogenous endothelium-derived vasodilators, but little information is available on their release during hypothermia. This study was carried out to test the hypothesis that endothelium may modulate vascular reactivity to decreased temperature changes. Segments of contracted (prostaglandin F(2alpha), 2x10(-6)M) canine coronary, femoral, and renal arteries, with and without endothelium, were in vitro ("organ chambers") exposed to progressive hypothermia (from 37 to 10 degrees C) in graded steps. The study is limited to physiological measurements of vascular tone, in the presence or absence of PGI(2) and/or NOS inhibitors, which show correlation with the relaxation. Hypothermia induced vasodilatation of vessels with intact endothelium, which became endothelium-independent below 20 degrees C. This vasodilatation began at 35 degrees C and, in the presence of indomethacin (2x10(-6)M), at 30 degrees C. Endothelium-dependent vasodilatation to hypothermia was blocked by L-NMMA or L-NOARG (10(-5)M), two competitive inhibitors of nitric oxide synthase (n=5 each, P<0.05). Oxyhemoglobin (2x10(-6)M) also inhibited vasodilatation induced by hypothermia (n=6, P<0.05). Pretreatment with either atropine or pirenzepine (10(-6)M) inhibited hypothermia-mediated vasodilatation (n=5 each, P<0.05). The present in vitro study concluded that the endothelium is sensitive to temperature variations and indicated that PGI(2) and NO-dependent pathways may be involved endothelium-dependent relaxation to hypothermia. The endothelium-dependent vasodilatation to hypothermia, in systemic and coronary arteries, is mediated by the M1 muscarinic receptor.     

Functional geometry of the permeation pathway of Ca2+-activated Cl-channels inferred from analysis of voltage-dependent block

We examined the voltage-dependent block of Ca(2+)-activated Cl(-) channels by anthacene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and niflumic acid (NFA) in excised inside-out and outside-out patches from Xenopus oocytes. The fraction of the voltage field (delta) experienced by the blocking drug was determined from the voltage dependence of block. All the drugs blocked by entering the channel from the outside. delta was 0.6 for A9C, 0.3 for DPC and DIDS, and <0.1 for NFA. Because the voltage dependence of the drugs differed, the order of potency was also voltage-dependent. At +100 mV the order of potency was NFA > A9C > DIDS > DPC (K(i) (microm) = 10.1, 18.3, 48, and 111, respectively). Because the drugs are hydrophobic, they can cross the bilayer when applied from the inside and block the channel from the outside. The equilibrium geometries of the blockers were determined by molecular modeling and compared with their blocking positions (delta). This analysis suggests that the channel is an elliptical cone with the largest opening facing the extracellular space. The selectivity filter has an apparent size of 0.33 x 0.75 nm, because C(CN)(3)-, which has these dimensions, permeates. The external opening is at least 0.60 x 0.94 nm, because DPC has these dimensions and penetrates the channel approximately 30%.     

CART peptide: central mediator of leptin-induced adipose tissue apoptosis?

Because of connections between CART peptide containing neurons and the sympathetic nervous system (SNS) and the possible role of the SNS in leptin-induced adipose apoptosis, CART may act as a downstream effector of leptin-induced adipose apoptosis. Male Sprague-Dawley rats received continuous intracerebroventricular (i.c.v.) infusion for 4 days of either artificial cerebrospinal fluid (aCSF, 12 microl/day), leptin (15 microg/day), or CART55-102 at 2.4 microg/day (CART2.4) or 9.6 microg/day (CART9.6). Food intake (FI) was decreased 10.8% for CART2.4, 41.9% for CART9.6 and 33.4% for leptin (p<0.05). CART9.6 and leptin reduced meal size and meal number. Body weight (BW) was reduced by CART9.6 (14.6%) and leptin (11.6%) (p<0.05), but not by CART2.4. CART9.6 and CART2.4, but not leptin, caused hypothermia, and CART9.6 inhibited physical activity (p<0.05). Epididymal, inguinal and retroperitoneal fat pad weights were reduced (p<0.05) by both CART treatments and leptin; CART9.6 also reduced gastrocnemius muscle weight (18.1%, p<0.05). Leptin, but not CART, increased serum free fatty acid concentrations by 31.1% (p<0.05) and increased adipose apoptosis by 48% (p<0.05). These data show that although leptin and CART55-102 have some similar actions, CART55-102 is probably not a mediator for leptin-induced adipose apoptosis in the brain.     

CNS melanocortin and leptin effects on stearoyl-CoA desaturase-1 and resistin expression

The objective of this study was to determine whether centrally administered leptin decreased liver and adipose SCD1 expression or adipose resistin expression, and whether these effects were mediated by central melanocortin receptors. Male Sprague-Dawley rats were injected into the lateral cerebral ventricle (i.c.v.) once daily for 4 days with artificial cerebrospinal fluid (aCSF, 5 microl), leptin (10 microg) or MTII (0.1 nmol); two other groups were pretreated icv with the melanocortin antagonist, SHU9119 (1.0 nmol), followed by leptin or MTII. Epididymal and inguinal adipose tissue and liver were collected after rats were killed and mRNA expression of SCD1 and resistin was measured. Both leptin and MTII reduced SCD1 expression and pretreatment with SHU9119 reversed their effects. Neither leptin nor MTII affected resistin expression, but it was increased by SHU9119. These results show that CNS melanocortin receptors are likely mediators of leptin's effects on SCD1 expression in liver and adipose tissue, The findings were inconclusive concerning the effects of leptin and melanocortins on adipose resistin expression.     

Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.     

Myostatin knockout in mice increases myogenesis and decreases adipogenesis

Growth differentiation factor-8 (GDF-8), or Myostatin, plays an important inhibitory role during muscle development. Since muscle and adipose tissue develop from the same mesenchymal stem cells, we hypothesized that Myostatin gene knockout may cause a switch between myogenesis and adipogenesis. Male and female wild type (WT) and Myostatin knockout (KO) mice were sacrificed at 4, 8, and 12 weeks of age. The gluteus muscle (GM) was larger in KO mice compared to WT mice at 8 (P < 0.01) and 12 (P < 0.001) weeks. At 12 weeks, KO mice had decreased fat depots (P < 0.01). Compared to 12-week-old WT mice, serum leptin concentration in KO mice was lower (P < 0.001) and leptin mRNA expression was decreased (P < 0.01) in inguinal adipose tissue. CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor-gamma (PPARgamma) levels in adipose tissue were significantly lower in KO mice compared to WT mice. Thus, increased muscle development in Myostatin knockout mice is associated with reduced adipogenesis and consequently, decreased leptin secretion.