Considerations in the development of biomarkers of copper status.

Copper (Cu) is an essential nutrient, but a harmful metal in excess. As part of the North American Dietary Reference Intakes, the Recommended Dietary Allowance for Cu was set using a combination of biomarkers of Cu deficiency, including plasma Cu and ceruloplasmin concentrations, erythrocyte Cu/Zn superoxide dismutase activity, and platelet Cu concentration. Liver damage was the sole indicator used in setting the Tolerable Upper Intake Level. Some studies suggest that these conventional biomarkers may not be sensitive enough to detect marginal reductions or excesses of Cu that could pose a health risk. The insensitivity of conventional biomarkers casts uncertainty as to the prevalence of Cu deficiency or overload in the population and in the accuracy of current nutritional reference values for Cu. Numerous biochemical changes have been associated with alterations in Cu status, and many potential biomarkers of Cu nutriture have been proposed; yet, conventional biomarkers are still the most frequently used, underscoring the need for research efforts to substantiate the use of novel biomarkers. In this report, biomarkers of Cu status are reviewed and practical considerations in the development of novel biomarkers are discussed as diagnostic tools for assessing Cu status in humans.

Regions of the catalytic alpha subunit of Na,K-ATPase important for functional interactions with FXYD 2.

The gamma modulator (FXYD 2) is a member of the FXYD family of single transmembrane proteins that modulate the kinetic behavior of Na,K-ATPase. This study concerns the identification of regions in the alpha subunit that are important for its functional interaction with gamma. An important effect of gamma is to increase K+ antagonism of cytoplasmic Na+ activation apparent as an increase in KNa' at high [K+]. We show that although gamma associates with alpha1, alpha2, and alpha3 isoforms, it increases the KNa' of alpha1 and alpha3 but not alpha2. Accordingly, chimeras of alpha1 and alpha2 were used to identify regions of alpha critical for the increased KNa'. As with alpha1 and alpha2, all chimeras associate with gamma. Kinetic analysis of alpha2front/alpha1back chimeras indicate that the C-terminal (Lys907-Tyr1018) region of alpha1, which includes transmembrane (TM)9 close to gamma, is important for the increase in KNa'. However, similar experiments with alpha1front/alpha2back chimeras indicate a modulatory role of the loop between TMs 7 and 8. Thus, as long as the alpha1 L7/8 loop is present, replacement of TM9 of alpha1 with that of alpha2 does not abrogate the gamma effect on KNa'. In contrast, as long as TM9 is that of alpha1, replacement of L7/8 of alpha1 with that of alpha2 does not abolish the effect. It is suggested that structural association of the TM regions of alpha and FXYD 2 is not the sole determinant of this effect of FXYD on KNa' but is subject to long range modulation by the extramembranous L7/8 loop of alpha.

Modulation of Na,K-ATPase by the gamma subunit: studies with transfected cells and transmembrane mimetic peptides.

The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the gamma subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.

Structure/function studies of the gamma subunit of the Na,K-ATPase.

The Na,K-ATPase gamma subunit is present primarily in kidney as two splice variants, gammaa and gammab, which differ only at their extracellular N-termini. Two distinct effects of gamma are seen in biochemical Na,K-ATPase assays of mammalian (HeLa) cells transfected with gammaa or gammab, namely, (i) a decrease in K'(ATP) probably secondary to a shift in steady-state E(1) <--> E(2) poise in favor of E(1) and (ii) an increase in cytoplasmic K(+)/Na(+) antagonism seen as an increase in K'(Na) at high K(+) concentration. Mutagenesis experiments involving alterations in extramembranous regions of gamma indicate that different regions mediate the aforementioned distinct effects and that the effects appear to be long range. Studies of ouabain-sensitive fluxes with intact cells confirm the gamma effects seen with membranes and also suggest an additional effect (increase) in apparent affinity for extracellular K(+). Alteration in gamma function was also evidenced in the behavior of a G41 -->R mutation within the transmembrane domain of gamma. G41R is associated with autosomal dominant renal magnesium wasting. Our studies show that this mutation in the gammab variant retards trafficking of gamma, but not alphabeta pumps, to the cell surface and abolishes functional effects of gamma, consistent with the conclusion that the Mg(2+) transport defect is secondary to loss of gamma modulation of Na,K-ATPase function.

MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence.

The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum-sensing mechanism in addition to nutritional signals. However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA. In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively. The metR gene was cloned based on a general strategy to detect lux DNA-binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant. Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP. Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor. Disruption of crp decreased luminescence by about 1,000-fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10-fold, confirming its function as a repressor. Comparison of the levels of the autoinducers involved in quorum sensing excreted by V. harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing. Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.