EPR and ENDOR Investigation of Rhodosemiquinone in Bacterial Reaction Centers Formed by B-Branch Electron Transfer.

In photosynthetic bacteria, light-induced electron transfer takes place in a protein called the reaction center (RC) leading to the reduction of a bound ubiquinone molecule, Q(B), coupled with proton binding from solution. We used electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) to study the magnetic properties of the protonated semiquinone, an intermediate proposed to play a role in proton coupled electron transfer to Q(B). To stabilize the protonated semiquinone state, we used a ubiquinone derivative, rhodoquinone, which as a semiquinone is more easily protonated than ubisemiquinone. To reduce this low-potential quinone we used mutant RCs modified to directly reduce the quinone in the Q(B) site via B-branch electron transfer (Paddock et al. in Biochemistry 44:6920-6928, 2005). EPR and ENDOR signals were observed upon illumination of mutant RCs in the presence of rhodoquinone. The EPR signals had g values characteristic of rhodosemiquinone (g(x) = 2.0057, g(y) = 2.0048, g(z) ∼ 2.0018) at pH 9.5 and were changed at pH 4.5. The ENDOR spectrum showed couplings due to solvent exchangeable protons typical of hydrogen bonds similar to, but different from, those found for ubisemiquinone. This approach should be useful in future magnetic resonance studies of the protonated semiquinone.

Valeriana officinalis root extracts have potent anxiolytic effects in laboratory rats.

Valerian root (Valeriana officinalis) is a popular and widely available herbal supplement, primarily used to treat insomnia and anxiety. Until recently, its mechanism of action has remained unknown. Neurobiological research has begun to show that the herb, with its active valerenic acid, interacts with the GABA(A)-ergic system, a mechanism of action similar to the benzodiazepine drugs. This series of experiments sought to corroborate these findings with behavioral measures, compare them to the benzodiazepine diazepam, and to analyze the chemical composition of Valeriana officinalis. Rats were administered either ethanol (1 ml/kg), diazepam (1mg/kg), valerian root extract (3 ml/kg), valerenic acid (3mg/kg), or a solution of valerenic acid and exogenous GABA (75 microg/kg and 3.6 microg/kg, respectively) and assessed for the number of entries and time spent on the open arms of an elevated plus maze. Results showed that there was a significant reduction in anxious behavior when valerian extract or valerenic acid exposed subjects were compared to the ethanol control group. The evidence supports Valeriana officinalis as a potential alternative to the traditional anxiolytics as measured by the elevated plus maze.

Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalyze both O-methyltransferase steps in coenzyme Q biosynthesis.

Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1-coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3, 4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to the Escherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q(3) precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that one O-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria.

Human carbonic anhydrase I concentration in erythrocytes during pregnancy and the menstrual cycle.

Carbonic anhydrase I (HCAI) concentrations were measured in erythrocytes (RBC) taken from nine women at intervals throughout their pregnancies and from thirteen normal women during the menstrual cycle. No significant changes in RBC HCAI concentrations were found in any instance. Small changes which were found in RBC HCAI concentrations were within experimental error and did not correlate with any other measured parameter. Changes in RBC HCAI concentrations in six men over a four-week period were of similar order to that found in the thirteen normal women over a similar length of time.

Human erythrocyte carbonic anhydrase I concentrations in dried blood spots from normal and hypothyroid neonates and children.

The ELISA technique was used to assay carbonic anhydrase I (HCAI) in hemolysates prepared by the elution of dried blood samples from Guthrie cards. The ratio HCAI (mg)/hemoglobin (g) measured in blood samples eluted from Guthrie cards was not significantly different from that determined in aliquots of the same blood samples after storage as erythrocyte (RBC) lysates at -20 degrees C, provided that the dried blood was eluted within three weeks of collection. The normal neonatal mean (SD) RBC HCAI concentrations were 2.05 (1.01) and 1.82 (0.86) mg HCAI/g hemoglobin for females and males respectively. Erythrocyte HCAI concentrations gradually rose with age, approaching normal adult levels by 16 years. Blood from hypothyroid neonates and hypothyroid infants on treatment gave normal HCAI/hemoglobin ratios.

The measurement of human erythrocyte carbonic anhydrase I by the enzyme linked immunosorbent assay (ELISA).

A method is described for the assay of human erythrocyte (RBC) carbonic anhydrase I (HCAI) by the enzyme linked immunosorbent assay. The method was found to be a simple, reliable and precise technique and gave a mean C.V. for 40 samples, assayed in quadruplicate, of 3.32% and a range of 0.84-5.9. The mean erythrocyte HCAI value and standard deviation for 20 normal men and women were respectively 16.9 +/- 3.4 and 15.4 +/- 2.1 mg HCAI/g haemoglobin. The use of heparin as an anticoagulant interfered with the assay resulting in apparent HCAI concentrations as low as 60% of those obtained using EDTA.