Immunophotochemical analysis of mineralocortin by polyclonal antibodies against the native receptor from rat kidney

We have obtained a polyclonal antiserum by immunizing fawn Burgundy rabbits with the mineralocorticoid receptor (MCR) purified biochemically from rat kidneys. High titers of anti-MCR activity were obtained in radioimmunoassays within 3 weeks and increased with a booster shot. In Western blot analysis, the antibody revealed a major band of 94–98 kDa in renal cytosol from rat and beef kidneys. We also developed a fluorographic procedure where the MCR linked covalently to tritiated R-5020, following ultraviolet irradiation, gave imprints superimposable on the Western blot profile. The fluorographic pattern was specific since it was largely abolished in the presence of cold RU 26752 that is specific to MCR, or mineralocortin. The immune IgG precipitated rat renal MCR-[³H]RU 26752 complexes in a dose-dependent manner and also recognized MCR bound to the natural hormone aldosterone. During gel permeation chromatography on Sephacryl, the elution profile of [³H]RU 26752 shifted to high-molecular-weight regions in the presence of immune IgG. The receptor protein could be immunolocalized primarily to the principal cells of the collecting duct in rat kidney but the intercalated cells and glomeruli were not labeled, contrary to beef kidney where a uniform pattern of immunostaining was evident. These should permit large-scale purification of the MCR for detailed physicochemical studies and for screening of the MCR-positive tissues during various pathophysiological syndromes.     

Paradoxical effects of mineralocorticoids on the ion gated sodium channel in embryologically diverse cells

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone. In quantitative PCR, both basal and aldosterone-induced levels of ENaC were diminished by ZK 91587 in the corneal fibroblast, in contrast to the stimulation observed in the retinal pigmentary epithelium. Thus, contrary to the existing notions, (a) antimineralocorticoids can act both as agonists and antagonists, and (b) the receptor-mediated action of mineralocorticoids on the sodium channel is not restricted to the epithelial cell.     

Mineralocorticoid hormone action in plant cells.

The multiplication of Chlamydomonas reinhardtii wild type cells can be arrested by the spirolactone RU 26752 and this is fully reversible by the natural mineralocorticoid aldosterone. Evidence is presented for a 52 kDa protein that possesses functional DNA and ligand binding domains and tests positive for mineralocorticoid receptor-like activity by immuneprecipitation, macroaggregation, and photoaffinity. The regulation of trans-activation by steroid hormones in the animal world would therefore appear to be just as valid for the plant kingdom, thereby providing a new model for genetic analysis.     

Two genetically distinct methyl-coenzyme M reductases in Methanobacterium thermoautotrophicum strain Marburg and delta H

Methyl-coenzyme M reductase (MCR) catalyzes the methane-forming step in methanogenic archaebacteria. The reductase has been characterized in detail from Methanobacterium thermoautotrophicum strain Marburg and delta H, which grow on H2 and CO2 as energy source. During purification of the enzyme we have now discovered a second methyl-coenzyme M reductase (MCR II) in the two strains, which elutes at lower salt concentration from anion-exchange columns than the enzyme (MCR I) previously characterized. MCR II is similar to MCR I in that it is also composed of three different subunits alpha, beta, and gamma but distinct from MCR I in that the gamma subunit is 5 kDa smaller, as revealed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of the alpha, beta, and gamma subunits of MCR II and MCR I were found to be different in several amino acid positions. The respective sequences showed, however, strong similarities indicating that MCR II was not derived from MCR I by limited proteolysis. The relative amounts of MCR I and MCR II present in the cells were affected by the growth conditions. When the cultures were supplied with sufficient H2 and and CO2 and the cells grew exponentially, essentially only MCR II was found. When growth was limited by the gas supply, MCR I predominated.     

Sulfhydryl groups are involved in the binding of agonists and antagonists to the human mineralocorticoid receptor

To investigate the role of sulfhydryl groups in the interaction of agonists and antagonists with the human mineralocorticoid receptor (hMR) the effect of methyl methanethiosulfonate (MMTS) on free and liganded-hMR was examined. hMR was expressed in insect cells (Sf9) using the baculovirus system. Treatment of cytosol with MMTS at 4°C inhibited the binding to hMR of both [³H]aldosterone and [³H]RU26752 (a synthetic aldosterone antagonist). At 4°C, the sensitivity to MMTS of the liganded-hMR complexes was dependent upon the nature of the ligands: agonists (aldosterone, corticosterone and cortisol) rendered the hMR resistant to MMTS, whereas antagonists (progesterone and RU26752) did not protect the receptor against MMTS inactivation. Analysis of the dose- and time-dependent effects of MMTS revealed that the free hMR and the RU26752-hMR complexes displayed a similar sensitivity to MMTS and that MMTS increased the dissociation of RU26752 from the hMR. At 4°C the aldosterone-hMR complexes were not affected by MMTS treatment, whereas at 20°C MMTS increased the dissociation of aldosterone from hMR. This effect was unrelated to the dissociation of hsp90 from hMR, because the sensitivity of the aldosterone-hMR complexes to MMTS remained unchanged after covalent linkage between hsp90 and the receptor. Our results suggest that agonists and antagonists modify the receptor conformation in distinct ways that render cysteine residues of the ligand binding domain more or less accessible to the MMTS action.     

Paradoxical differences in the receptor binding of two new antimineralocorticoids

Two synthetic derivatives of spironolactone were used to examine various aspects of the mineralocorticoid receptor structure and function. Introduction of a propyl residue in the 7-position of spironolactone produced a molecule (RU 26752) that saturated the aldosterone specific receptor in the 1-10 nM range, and another, more abundant species in the 10-100 nM range which had little affinity for the natural hormone. The specificity for both sites was increased when the methoxycarbonyl group was introduced in the 7-position (ZK 91587). Neither antagonist exhibited affinity for blood serum transcortin or receptors in non-target organs like the lung and the liver. RU 26752-receptor complex was more unstable than the hormone-receptor complex at 35 degrees C but underwent comparable thermal activation as evidenced by binding to DNA cellulose and the 7 S to 4 S shift on sucrose gradients. In contrast, ZK 91587 did not permit thermal activation and greatly labilized the receptor at 35 degrees C. In ion exchange chromatography, two peaks were observed with unactivated ZK 91587-receptor complex, but RU 26752 was bound exclusively to the component eluted with high salt. Molecular filtration revealed two peaks of bound radioactivity with both antimineralocorticoids. These studies reveal important differences in the mechanism of action of two antagonists differing solely in the residue in position 7 of the spironolactone molecule. Such differences could be exploited to purify the mineralocorticoid receptor and clinically to prescribe the appropriate drug with greater precision.     

Production and metabolic clearance rates of 1,25-dihydroxyvitamin D3 during maturation in rats: studies using a rapid, primed-infusion technique

I have adapted the primed-infusion technique for the rapid estimation of the metabolic clearance rate (MCR) and production rate (PR) of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in chronically catheterized conscious rats. Following a bolus injection, [3H]-1,25(OH)2D3 was infused iv at a constant rate for 7 h. Steady-state [3H]-1,25(OH)2D3 levels in plasma were achieved within 3 h. HPLC-purification of plasma [3H]-1,25(OH)2D3 was necessary. This rapid, primed-infusion technique thus eliminates the need for protracted infusions to achieve steady-state plasma [3H]-1,25(OH)2D3 levels. The MCR averaged 0.201 +/- 0.003 ml.min-1.kg-1 in fed male rats weighing 200-300 g. This MCR is approximately 50% lower than that seen in other species. Overnight fasting was without effect on the MCR. The MCR increased in direct proportion to body weight in maturing rats (6-16 weeks old) weighing 150-450 g. Thus, the MCR can be normalized per kg across this age range. However, 1,25(OH)2D3 production and plasma levels both decreased by 65-67% as the rats matured. The failure of a 3-fold decrease in plasma 1,25(OH)2D3 levels to affect the MCR suggests that 1,25(OH)2D3 stimulates its own catabolism only at markedly elevated concentrations.     

Analysis of the mineralocorticoid receptor in rat heart with the aid of two new spirolactone derivatives

Two derivatives of spirolactone, synthesized in an effort to eliminate the obnoxious side effects of the native molecule, were employed to dissect various aspects of the MR structure and function in rat heart. The introduction of a propyl residue in position 7 of spirolactone produced a molecule (RU 26752) that exhibited an increased affinity for the agonist specific MR, and furthermore revealed an antagonist-specific MR population in the target organ heart but absent from nontarget lung and liver. The specificity for both sites increased when a methoxycarbonyl group was introduced in the 7 position (ZK 91587). RU 26752 labilized the MR at 35 degrees C but did not interfere with thermal activation assessed on DNA-cellulose and sucrose density gradients. ZK 91587 was even more effective in labilizing the MR and did not permit thermal activation at all. Whereas only one ionic species was observed with RU 26752 on DE-52 columns, two were evident with ZK 91587. Both antimineralocorticoids were bound to populations of two molecular sizes on Ultrogel columns. Thus, the nature of chemical substitution in the 7 position of spirolactone dramatically alters the receptor-mediated antisteroid action of the resulting molecule. Such differences may permit distinction between agonist versus the antagonist-specific receptor conformations, and could possibly be exploited for the eventual purification of the mineralocorticoid receptor from various organs.     

The early non-genomic aldosterone-induced increase in sodium transport is a membrane-initiated event that requires protein carboxyl methylation in renal cells.

Effects of aldosterone on its target cells are generally considered to be mediated through the genomic pathway. However, recent studies have evidenced rapid effects of the hormone that involve a non-genomic mechanism. In this study, we show that, in the RCCD2 rat cortical collecting duct cell line, the early effect of the hormone on transepithelial sodium transport is neither antagonized by the mineralo- and glucocorticoid receptors antagonists RU26752 and RU486, nor blocked by mRNA and protein synthesis inhibitors. Interestingly, the plasma membranes of RCCD2 cells specifically bind 3H-aldosterone but not 3H-dexamethasone, a binding that is not displaced in the presence of RU26752 or RU486, suggesting the presence of an aldosterone membrane receptor. In addition, the early aldosterone-induced increase in sodium transport is blocked by the addition of a specific inhibitor of carboxyl methyl transferase. These results suggest that, in RCCD2 cells, the early aldosterone-induced increase in sodium transport is not mediated through the genomic pathway but through a membrane receptor-mediated signal and could involve a rapid carboxyl methylation process regulated by aldosterone.     

Comparison of three methyl-coenzyme M reductases from phylogenetically distant organisms: unusual amino acid modification, conservation and adaptation

The nickel enzyme methyl-coenzyme M reductase (MCR) catalyzes the terminal step of methane formation in the energy metabolism of all methanogenic archaea. In this reaction methyl-coenzyme M and coenzyme B are converted to methane and the heterodisulfide of coenzyme M and coenzyme B. The crystal structures of methyl-coenzyme M reductase from Methanosarcina barkeri (growth temperature optimum, 37 degrees C) and Methanopyrus kandleri (growth temperature optimum, 98 degrees C) were determined and compared with the known structure of MCR from Methanobacterium thermoautotrophicum (growth temperature optimum, 65 degrees C). The active sites of MCR from M. barkeri and M. kandleri were almost identical to that of M. thermoautotrophicum and predominantly occupied by coenzyme M and coenzyme B. The electron density at 1.6 A resolution of the M. barkeri enzyme revealed that four of the five modified amino acid residues of MCR from M. thermoautotrophicum, namely a thiopeptide, an S-methylcysteine, a 1-N-methylhistidine and a 5-methylarginine were also present. Analysis of the environment of the unusual amino acid residues near the active site indicates that some of the modifications may be required for the enzyme to be catalytically effective. In M. thermoautotrophicum and M. kandleri high temperature adaptation is coupled with increasing intracellular concentrations of lyotropic salts. This was reflected in a higher fraction of glutamate residues at the protein surface of the thermophilic enzymes adapted to high intracellular salt concentrations.     

Biochemical characterization of metabolism‐based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F₂ population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F₂ population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.     

Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes

Hepatocytes prepared from 18-day-old mouse embryos were grown in serum-free medium and reached confluence after two days in culture. The total amount of the 26 kDa gap junction protein decreased in these cells during the first 24 h in culture and increased again between day 1 and day 3 more than 10-fold. At day 3 a half-life time of 2.5 to 3 h was determined for the 26 kDa protein by [35S]methionine incorporation and immunoprecipitation using affinity-purified anti-26 kDa. Incorporation of [32P]orthophosphate into the 26 kDa protein of cultured hepatocytes was found at serine residues (98%) and tyrosine residues (about 2%). The addition of dibutyryl cyclic adenosine monophosphate (db cAMP) to the culture medium at day 2 had two effects: After 15 min the extent of phosphorylation of the 26 kDa protein increased 2.7-fold whereas the total amount of the 26 kDa protein increased only 1.2-fold. After 3 h of incubation with db cAMP, a 2.5-fold increase of the 26 kDa protein was noticed which was accompanied by a 3.2-fold increase in phosphorylation of serine residues. The effects of db cAMP on phosphorylation of the 26 kDa protein could be augmented or mimicked by the addition of isoproterenol, theophylline or forskolin to the culture medium of hepatocytes. In extracts of rat hepatocarcinoma MH1C1 cells and dog kidney MDCK cells, a phosphorylated 26 kDa protein can be immunoprecipitated using anti-liver 26 kDa. These results demonstrate that the gap junction 26 kDa protein can be posttranslationally modified by cAMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic and kinetic studies of the reaction of bromopropanesulfonate with methyl-coenzyme M reductase

Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.     

The ubiquitin-like protein Smt3p is activated for conjugation to other proteins by an Aos1p/Uba2p heterodimer.

SMT3 is an essential Saccharomyces cerevisiae gene encoding a 11.5 kDa protein similar to the mammalian ubiquitin-like protein SUMO-1. We have found that Smt3p, like SUMO-1 and ubiquitin, can be attached to other proteins post-translationally and have characterized the processes leading to the activation of the Smt3p C-terminus for conjugation. First, the SMT3 translation product is cleaved endoproteolytically to expose Gly98, the mature C-terminus. The presence of Gly98 is critical for Smt3p's abilities to be conjugated to protein substrates and to complement the lethality of a smt3Delta strain. Smt3p undergoes ATP-dependent activation by a novel heterodimeric enzyme consisting of Uba2p, a previously identified 71 kDa protein similar to the C-terminus of ubiquitin-activating enzymes (E1s), and Aos1p (activation of Smt3p), a 40 kDa protein similar to the N-terminus of E1s. Experiments with conditional uba2 mutants showed that Uba2p is required for Smt3p conjugation in vivo. Furthermore, UBA2 and AOS1 are both essential genes, providing additional evidence that they act in a distinct pathway whose role in cell viability is to conjugate Smt3p to other proteins.     

Possible errors of the Mutual Climatic Range (MCR) method in reconstructing the Pleistocene climate of Beringia

The main causes and degree of inaccuracy of air temperature reconstruction by the MCR method are discussed. This inaccuracy appears to result from several reasons: insufficient knowledge of geographic ranges of species, the use of calibration equations calculated for a different region, and the absence of generally accepted criteria for the selection of species to be used in MCR analysis, in relation to their distribution and ecological specificity. The maximum deviation of the reconstructed July temperature from the actual one was 2–4°C, which was 2–3 times the accepted MCR standard error. The requirements for selection of fossil species in order to improve the reconstruction accuracy are discussed.     

Movable computer ruler (MCR): a new method for measuring the size of Toxoplasma gondii cysts, tachyzoites and other selected parasites

A new method for measuring the size of parasites and other objects using optical microscopy was developed using a specifically designed movable computer ruler (MCR) derived from digital images of a stage micrometer. Subsequently, MCR can be superimposed on images of parasites to measure their size. MCR derived from the stage micrometer under a particular objective lens can be used to measure the size of an object acquired by the same lens/microscope/camera system. The conditions are fixed for every superimposed image including width, height, pixel number and density. The MCR was tested using selected parasites, and shown to be as accurate as the ocular micrometer disk, screw micrometer eyepiece and image analysis software. The lower technical complexity of the MCR method makes it applicable even in laboratories with limited resources.     

Effect of acute sympathetic nervous system activation on flow-mediated dilation of brachial artery

We tested the hypothesis that flow-mediated dilation (FMD) of the brachial artery would be impaired by acute increases in sympathetic nervous system activity (SNA) in models where similar peak shear stress stimulus was achieved by varying the duration of forearm muscle ischemia. Eleven healthy young men were studied under four different conditions, each with its own control: lower body suction (LBS), cold pressor test (CPT), mental arithmetic task (MAT), and activation of muscle chemoreflex (MCR). The duration of ischemia before observation of FMD by ultrasound imaging was 5 min each for control, LBS, and CPT; 3 min for MAT; and 2-min for MCR. Peak shear rate was not different between control and any of the SNA conditions, although total shear in the first minute was reduced in MAT. MCR was the only condition in which brachial artery vasoconstriction was observed before forearm occlusion [4.38 (SD 0.53) vs. control 4.60 (SD 0.53) mm, P < 0.05]; however, diameter increased to the same absolute value as that of the control, so the percent FMD was greater for MCR [9.85 (SD 2.33) vs. control 5.29 (SD 1.50)%]. Blunting of the FMD response occurred only in the CPT model [1.51 (SD 1.20)%]. During SNA, the increase in plasma cortisol from baseline was significant only for MCR; the increase in plasma norepinephrine was significant for MCR, LBS, and CPT; and the increase in epinephrine was significant only for MCR. These results showed that the four models employed to achieve increases in SNA had different effects on baseline brachial artery diameter and that blunted FMD is not a general response to increased SNA.     

Estradiol and testosterone metabolic clearance and production rates during puberal development in boars

Changes in metabolic clearance rate (MCR) and production rate (PR) of estradiol-17 beta (E2) and testosterone (T) were evaluated in crossbred boars averaging 35 +/- 1, 91 +/- 3, 118 +/- 4 and 177 +/- 6 kg at 80, 160, 260 and 560 days of age, respectively. A comparison of E2 and T MCR determined in blood or plasma was made in castrate and intact boars at 180 days of age. In the two experiments, estimates of MCR of E2 and T were consistently greater (22.0 and 23.8%) in blood than in plasma. These differences were not influenced by age of boar or castration. The MCR (l X day X kg BW) for E2 and T in plasma was greater (P less than 0.05) for 80-day-old prepuberal boars than the three groups of older boars. Production rates of E2 and T were lower in boars at 80 days of age than in older boars. Thus, a reduction in the MCR and an increase in PR of E2 and T in the boar are involved in the increased concentrations of circulating steroids associated with puberal development. Difference in MCR, determined in blood and plasma for both E2 and T, suggests that the contribution of the cellular component of blood to MCR studies in pigs should not be ignored.     

Activation of mineralocorticoid agonist and antagonist specific receptors from rat kidney

The kinetics of saturation, as well as of denaturation, confirm the existence of two distinct mineralocorticoid receptor populations one each for the agonist aldosterone (MR2) and the antagonist RU 26752 (MR3) in rat kidney. Receptor activation in vitro was dependent upon the buffer, progressed just as well in the presence of the agonist and the antagonist, and was inhibited by molybdate. These necessitate a reassessment of both the importance of receptor activation in vitro and its possible contribution to hormone action in vivo.