Modulation of NO binding to cytochrome <Emphasis Type=Italic>c</Emphasis>′ by distal and proximal haem pocket residues

We have cloned and expressed the cycP gene encoding cytochrome c′ from Alcaligenes xylosoxidans and generated mutations in Arg-124 and Phe-59, residues close to the haem, to probe their involvement in modulating the unusual spin-state equilibrium of the haem Fe and the unique proximal mode of binding of NO to form a stable five-coordinate adduct. Arg-124 is located in the proximal pocket of the haem and forms a hydrogen bond to the stable five-coordinated bound NO. Phe-59 provides steric hindrance at the distal face where NO binds initially to form a six-coordinate adduct. Optical spectroscopy showed altered electronic properties of the oxidised haem centre resulting from the mutations of both residues. The high affinity of the ferrous proteins for NO remained unchanged and all of the mutational variants formed a stable five-coordinate NO species (λ _Soret 395 nm) in the presence of stoichiometric concentrations of NO. However, the kinetics of the reactivity towards NO were altered, with mutation of the distal Phe-59 residue resulting in the transient six-coordinate distally bound NO adduct (λ _Soret 415 nm) not being detected. Surprisingly, substitution of the proximal residue Arg-124 with Phe, Ala, Gln or Glu also resulted in the six-coordinate adduct not being detected, showing that this proximal residue also modulates reactivity towards NO on the opposite haem face. In contrast, the R124L substitution retained the property of the native protein in the initial formation of a six-coordinate NO adduct, a finding of functional importance since a Lys or an Arg residue is invariant in these proteins.     

Expanded blood volumes contribute to the increased cardiovascular performance of endurance-trained older men

To determine whether expanded intravascular volumes contributeto the older athlete's higher exercise stroke volume and maximal oxygen consumption(O_{2 max}),we measured peak upright cycle ergometry cardiac volumes(^99mTc ventriculography) andplasma (¹²⁵I-labeled albumin) andred cell (NaCr⁵¹) volumes in 7 endurance-trained and 12 age-matched lean sedentary men. The athleteshad ~40% higherO_{2 max} values thandid the sedentary men and larger relative plasma (46 vs. 38 ml/kg), red cell (30 vs. 26 ml/kg), and total blood volumes (76 vs. 64 ml/kg) (allP < 0.05). Athletes hadlarger peak cycle ergometer exercise stroke volume indexes (75 vs. 57 ml/m²,P < 0.05) and 17% largerend-diastolic volume indexes. In the total group,O_{2 max}correlated with plasma, red cell, and total blood volumes(r = 0.61-0.70,P < 0.01). Peakexercise stroke volume was correlated directly with the blood volumevariables (r = 0.59-0.67,P < 0.01). Multiple regressionanalyses showed that fat-free mass and plasma or total blood volume,but not red cell volume, were independent determinants ofO_{2 max} andpeak exercise stroke volume. Plasma and total blood volumes correlated with the stroke volume and end-diastolic volume changes from rest topeak exercise. This suggests that expanded intravascular volumes, particularly plasma and total blood volumes, contribute to the higherpeak exercise left ventricular end-diastolic volume, stroke volume, andcardiac output and hence the higherO_{2 max} in master athletes by eliciting both chronic volume overload and increased utilization of the Frank-Starling effect during exercise.

     

Oleate-induced decrease in hepatocyte insulin binding is mediated by PKC-delta

We have previously shown that free fatty acids (FFA) impair hepatic insulin extraction in vivo and thus generate hyperinsulinemia, a suspected risk factor for atherosclerosis and cancer. Hepatic insulin extraction is a receptor-mediated event, which is initiated by hepatocyte insulin binding. In the present study, we investigated the effect of FFA on insulin binding in freshly isolated rat hepatocytes maintained at 10 mM glucose. Hepatocyte insulin binding decreased after 1 h exposure to oleate in a concentration-dependent manner reaching a maximum (35-40%) at 125 microM. Inhibition of FFA oxidation by >90% with the carnitine palmitoyltransferase I (CPT-I) inhibitor methylpalmoxirate (MP, 30 microM) did not prevent the effect of oleate. However, when hepatocytes were treated with the PKC inhibitor bisindolylmaleimide (BIM, 1 microM) the effect of oleate was abolished. Subcellular fractionation and immunoblotting of specific PKC isoforms revealed that oleate-induced hepatic PKC-delta membrane translocation, but did not translocate-epsilon, -theta, -alpha, -betaI and -betaII. These results indicate that PKC-delta activation mediated the FFA-induced decrease in hepatocyte insulin binding under our conditions, and thus provides a mechanistic basis for FFA-induced hyperinsulinemia.     

Adult obesity does not predict 6-year weight gain in men: the Aerobics Center Longitudinal Study

Objective: To determine the longitudinal relation between history of adult obesity and the 6‐year trajectory of weight change in men. Research Methods and Procedures: Subjects were healthy, affluent men (n = 761) between the ages of 20 and 78 years who completed at least four comprehensive medical exams at the Cooper Clinic between 1987 and 2003. Maximum adult weight was reported, and current height was measured at baseline. Body weight and cardiorespiratory fitness were measured at all examinations. Adult obesity status was determined from self‐reported maximum weight and measured height at baseline as BMI ≥ 30 kg/m². Weight at all examinations was regressed on a history of adult obesity using linear mixed effects modeling. Results: At baseline, men reporting a history of adult obesity were significantly heavier than men reporting no such history (BMI 29.8 vs. 25.0 kg/m²; p < 0.05). However, the rate of weight gain among men with a history of obesity was slower than among men without a history of adult obesity (0.04 vs. 0.18 kg/yr; p = 0.09), although this difference was only marginally significant. Fitness modulated the relationship between history of obesity and weight change over time, and both higher levels of fitness and greater frequency of dieting were associated with attenuated weight gain. In contrast, chronic disease and depression were associated with accelerated weight gain. Discussion: Although a history of obesity was associated with higher weight, it did not seem to result in accelerated weight gain over time. Additionally, dieting and fitness were important for minimizing weight gain.     

"Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease, RNase E

Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.