H+/2e- stoichiometry of the NADH : ubiquinone reductase reaction catalyzed by submitochondrial particles

Nitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio (H) over right arrow (+)/2e(-) (n) for the coupled reaction of NADH oxidation by the quinone accepters. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q(1) gives the value of n = 4. Thermally induced deactivation of Complex I [1, 2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q(1)-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q(1)-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex 1 as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme.

Resolving candidate genes of mouse skeletal muscle QTL via RNA-Seq and expression network analyses

BACKGROUND:

We have recently identified a number of Quantitative Trait Loci (QTL) contributing to the 2-fold muscle weight difference between the LG/J and SM/J mouse strains and refined their confidence intervals. To facilitate nomination of the candidate genes responsible for these differences we examined the transcriptome of the tibialis anterior (TA) muscle of each strain by RNA-Seq.

13,726 genes were expressed in mouse skeletal muscle. Intersection of a set of 1061 differentially expressed transcripts with a mouse muscle Bayesian Network identified a coherent set of differentially expressed genes that we term the LG/J and SM/J Regulatory Network (LSRN). The integration of the QTL, transcriptome and the network analyses identified eight key drivers of the LSRN (Kdr, Plbd1, Mgp, Fah, Prss23, 2310014F06Rik, Grtp1, Stk10) residing within five QTL regions, which were either polymorphic or differentially expressed between the two strains and are strong candidates for quantitative trait genes (QTGs) underlying muscle mass. The insight gained from network analysis including the ability to make testable predictions is illustrated by annotating the LSRN with knowledge-based signatures and showing that the SM/J state of the network corresponds to a more oxidative state. We validated this prediction by NADH tetrazolium reductase staining in the TA muscle revealing higher oxidative potential of the SM/J compared to the LG/J strain (p<0.03).

Thus, integration of fine resolution QTL mapping, RNA-Seq transcriptome information and mouse muscle Bayesian Network analysis provides a novel and unbiased strategy for nomination of muscle QTGs.

Familial idiopathic methemoglobinemia revisited: original cases reveal 2 novel mutations in NADH cytochrome b5 reductase

In 1943, the first description of familial idiopathic methemoglobinemia in the United Kingdom was reported in 2 members of one family. Five years later, Quentin Gibson (then of Queen's University, Belfast, Ireland) correctly identified the pathway involved in the reduction of methemoglobin in the family, thereby describing the first hereditary trait involving a specific enzyme deficiency. Recessive congenital methemoglobinemia (RCM) is caused by a deficiency of reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase. One of the original propositi with the type 1 disorder has now been traced. He was found to be a compound heterozygote harboring 2 previously undescribed mutations in exon 9, a point mutation Gly873Ala predicting a Gly291Asp substitution, and a 3-bp in-frame deletion of codon 255 (GAG), predicting loss of glutamic acid. A brother and a surviving sister are heterozygous; each bears one of the mutations. Thirty-three different mutations have now been recorded for RCM. The original authors' optimism that RCM would provide material for future genetic studies has been amply justified.

Recessive congenital methaemoglobinaemia: cytochrome b(5) reductase deficiency

Some 60 years ago, Quentin Gibson reported the first hereditary disorder involving an enzyme when he deduced that familial methaemoglobinaemia was caused by an enzymatic lesion associated with the glycolysis pathway in red blood cells. This disorder, now known as recessive congenital methaemoglobinaemia (RCM), is caused by NADH-cytochrome b5 reductase (cb(5)r) deficiency. Two distinct clinical forms, types I and II, have been recognized, both characterized by cyanosis from birth. In type II, the cyanosis is accompanied by neurological impairment and reduced life expectancy. Cytochrome b(5) reductase is composed of one FAD and one NADH binding domain linked by a hinge region. It is encoded by the CYB5R3 (previously known as DIA1) gene and more than 40 mutations have been described, some of which are common to both types of RCM. Mutations associated with type II tend to cause incorrect splicing, disruption of the active site or truncation of the protein. At present the description of the sequence variants of cb(5)r in the literature is confusing, due to the use of two conventions which differ by one codon position. Herein we propose a new system for nomenclature of cb(5)r based on recommendations of the Human Genome Variation Society. The development of a heterologous expression system has allowed the impact of naturally occurring variants of cb(5)r to be assessed and has provided insight into the function of cb(5)r.

Family-based investigation of the C677T polymorphism of the methylenetetrahydrofolate reductase gene in ischaemic heart disease

Background: Elevated homocysteine is associated with ischaemic heart disease (IHD). The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene results in reduced MTHFR enzyme activity and reduced methylation of homocysteine to methionine resulting in mild hyperhomocysteinaemia. Case-control association studies of the role of the C677T MTHFR polymorphism in IHD have produced conflicting results. We therefore used newly described family-based association tests to investigate the role of this polymorphism in IHD, in a well-defined population. Methods: A total of 352 individuals from 129 families (discordant sibships and parent-child trios) were recruited. Linkage disequilibrium between the polymorphism and IHD was tested for using the combined transmission disequilibrium test (TDT)/sib-TDT and pedigree disequilibrium test (PDT). Homocysteine levels were measured. Results: Both the TDT/sib-TDT and PDT analyses found a significantly reduced transmission of the T allele to affected individuals (P=0.016 and P=0.021). There was no significant difference in homocysteine levels between affected and unaffected siblings. TT homozygotes had mean homocysteine levels significantly higher than those of TC heterozygotes (P

Impaired functioning of thermolabile methylenetetrahydrofolate reductase is dependent on riboflavin status: implications for riboflavin requirements.

BACKGROUND: Methylenetetrahydrofolate reductase (MTHFR; EC 1.7.99.5) supplies the folate needed for the metabolism of homocysteine. A reduction in MTHFR activity, as occurs in the homozygous state for the 677C-->T (so-called thermolabile) enzyme variant (TT genotype), is associated with an increase in plasma total homocysteine (tHcy). OBJECTIVE: In vitro studies suggest that the reduced activity of thermolabile MTHFR is due to the inappropriate loss of its riboflavin cofactor. We investigated the hypothesis that MTHFR activity in the TT genotype group is particularly sensitive to riboflavin status. DESIGN: We studied tHcy and relevant B-vitamin status by MTHFR genotype in a cross-sectional study of 286 healthy subjects aged 19-63 y (median: 27 y). The effect of riboflavin status was examined by dividing the sample into tertiles of erythrocyte glutathionine reductase activation coefficient, a functional index of riboflavin status. RESULTS: Lower red blood cell folate (P = 0.0001) and higher tHcy (P = 0.0082) concentrations were found in the TT group than in the heterozygous (CT) or wild-type (CC) groups. However, these expected relations in the total sample were driven by the TT group with the lowest riboflavin status, whose mean tHcy concentration (18.09 micromol/L) was almost twice that of the CC or CT group. By contrast, adequate riboflavin status rendered the TT group neutral with respect to tHcy metabolism. CONCLUSIONS: The high tHcy concentration typically associated with homozygosity for the 677C-->T variant of MTHFR occurs only with poor riboflavin status. This may have important implications for governments considering new fortification policies aimed at the prevention of diseases for which this genotype is associated with increased risk.

Nitrate reductase activity in macroalgae and its vertical distribution in macroalgal epiphytes of seagrasses.

Macroalgal epiphytes within seagrass meadows make a significant contribution to total primary production by assimilating water column N and transferring organic N to sediments. Assimilation of NO3 – requires nitrate reductase (NR, EC 1.6.6.1); NR activity represents the capacity for NO3 – assimilation. An optimised in vitro assay for determining NR activity in algal extracts was applied to a wide range of macroalgae and detected NR activity in all 22 species tested with activity 2 to 290 nmolNO3 – min–1 g–1 frozen thallus. With liquid-N2 freezing immediately after sample collection, this method was practical for estimating NR activity in field samples. Vertical distribution of NR activity in macroalgal epiphytes was compared in contrasting Posidonia sinuosa and Amphibolis antarctica seagrass meadows. Epiphytes on P. sinuosa had higher mass-specific NR activity than those on A. antarctica. In P. sinuosa canopies, NR activity increased with distance from the sediment surface and was negatively correlated with [NH4 +] in the water but uncorrelated with [NO3 –]. This supported the hypothesis that NH4 + released from the sediment suppresses NR in epiphytic algae. In contrast, the vertical variation in NR activity in macroalgae on A. antarctica was not statistically significant although there was a weak correlation with [NO3 –], which increased with distance from the sediment. Estimated capacities for NO3 – assimilation in macroalgae epiphytic on seagrasses during summer (24 and 46 mmolN m–2 d–1 for P. sinuosa and A. antarctica, respectively) were more than twice the estimated N assimilation rates in similar seagrasses. When the estimates were based on annual average epiphyte loads for seagrass meadows in other locations, they were comparable to those of seagrasses. We conclude that epiphytic algae represent a potentially important sink for water-column nitrate within seagrass meadows.