Characterisation of the redox centers of ethylbenzene dehydrogenase

AbstractEthylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI–MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH. Graphical abstract

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Differential Regulation of the Epithelial Sodium Channel (ENaC) in Rat Kidney, Lung and Distal Colon in Response to Aldosterone.

Hypertension ◽

10.1161/hyp.36.suppl_1.724 ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 724-724

Author(s):

Shyama M E Masilamani ◽

Gheun-Ho Kim ◽

Mark A Knepper

Keyword(s):

Sodium Channel ◽

Epithelial Sodium Channel ◽

Distal Colon ◽

Β Subunit ◽

Dietary Salt ◽

Α Subunit ◽

Salt Restriction ◽

Γ Subunit ◽

Dietary Salt Restriction ◽

Epithelial Sodium Channel Enac

P170 The mineralocorticoid hormone, aldosterone increases renal tubule Na absorption via increases in the protein abundances of the α-subunit of the epithelial sodium channel (ENaC) and the 70 kDa form of the γ- subunit of ENaC (JCI 104:R19-R23). This study assesses the affect of dietary salt restriction on the regulation of the epithelial sodium channel (ENaC) in the lung and distal colon, in addition to kidney, using semiquantitative immunoblotting. Rats were placed initially on either a control Na intake (0.02 meq/day), or a low Na intake (0.2 meq/day) for 10 days. The low salt treated rats demonstrated an increase in plasma aldosterone levels at day 10 (control = 0.78 + 0.32 nM; Na restricted = 3.50 + 1.30 nM). In kidney homogenates, there were marked increases in the band density of the α-subunit of ENaC (286 % of control) and the 70 kDa form of γ-subunit of ENaC (262 % of control), but no increase in the abundance of the β-subunit of ENaC. In lung homogenates, there was no significant change in the band densities of the α, β, or γ subunits of ENaC. In distal colon, there was an increase in the band density of the β-subunit of ENaC (311 % of control) and an increase in both the 85 kDa (2355% of control) and 70 kDa (843 % of control) form of the γ subunit of ENaC in response to dietary Na restriction. However, there was no significant difference in the band density of the α-subunit of ENaC. These findings demonstrate tissue specific regulation of the three subunits of ENaC in response to dietary salt restriction.

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Altered expression of renal NHE3, TSC, BSC-1, and ENaC subunits in potassium-depleted rats

AJP Renal Physiology ◽

10.1152/ajprenal.00186.2002 ◽

2002 ◽

Vol 283 (6) ◽

pp. F1376-F1388 ◽

Cited By ~ 45

Author(s):

Marie-Louise Elkjær ◽

Tae-Hwan Kwon ◽

Weidong Wang ◽

Jakob Nielsen ◽

Mark A. Knepper ◽

...

Keyword(s):

Sodium Channel ◽

Β Subunit ◽

Outer Medulla ◽

Α Subunit ◽

Altered Expression ◽

Γ Subunit ◽

Outer Stripe ◽

Type 3 ◽

Epithelial Sodium Channel Enac ◽

Concentrating Defect

The purpose of this study was to examine whether hypokalemia is associated with altered abundance of major renal Na+ transporters that may contribute to the development of urinary concentrating defects. We examined the changes in the abundance of the type 3 Na+/H+ exchanger (NHE3), Na+-K+-ATPase, the bumetanide-sensitive Na+-K+-2Cl− cotransporter (BSC-1), the thiazide-sensitive Na+-Cl− cotransporter (TSC), and epithelial sodium channel (ENaC) subunits in kidneys of hypokalemic rats. Semiquantitative immunoblotting revealed that the abundance of BSC-1 (57%) and TSC (46%) were profoundly decreased in the inner stripe of the outer medulla (ISOM) and cortex/outer stripe of the outer medulla (OSOM), respectively. These findings were confirmed by immunohistochemistry. Moreover, total kidney abundance of all ENaC subunits was significantly reduced in response to the hypokalemia: α-subunit (61%), β-subunit (41%), and γ-subunit (60%), and this was confirmed by immunohistochemistry. In contrast, the renal abundance of NHE3 in hypokalemic rats was dramatically increased in cortex/OSOM (736%) and ISOM (210%). Downregulation of BSC-1, TSC, and ENaC may contribute to the urinary concentrating defect, whereas upregulation of NHE3 may be compensatory to prevent urinary Na+ loss and/or to maintain intracellular pH levels.

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Trophectoderm development and function: the roles of Na+/K+-ATPase subunit isoforms

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y02-017 ◽

2002 ◽

Vol 80 (2) ◽

pp. 110-115 ◽

Cited By ~ 12

Author(s):

Gerald M Kidder

Keyword(s):

Fluid Transport ◽

Shape Change ◽

Basolateral Membrane ◽

Preimplantation Development ◽

Preimplantation Embryos ◽

Β Subunit ◽

Atpase Subunit ◽

Α Subunit ◽

Γ Subunit ◽

Subunit Isoforms

Preimplantation development is a period of cell division, cell shape change, and cell differentiation leading to the formation of an epithelium, the trophectoderm. The trophectoderm is the part of the conceptus that initiates uterine contact and, after transformation to become the trophoblast, uterine invasion. Thus, trophectoderm development during preimplantation stages is a necessary antecedent to the events of implantation. The preimplantation trophectoderm is a transporting epithelium with distinct apical and basolateral membrane domains that facilitate transepithelial Na+ and fluid transport for blastocoel formation. That transport is driven by Na+/K+-ATPase localized in basolateral membranes of the trophectoderm. Preimplantation embryos express multiple α and β subunit isoforms of Na+/K+-ATPase, potentially constituting multiple isozymes, but the basolaterally located α1β1 isozyme uniquely functions to drive fluid transport. They also express the γ subunit, which is a modulator of Na+/K+-ATPase activity. In the mouse, two splice variants of the γ subunit, γa and γb, are expressed in the trophectoderm. Antisense knockdown of γ subunit accumulation caused a delay of cavitation, implying an important role in trophectoderm function. The preimplantation trophectoderm offers a unique model for understanding the roles of Na+/K+-ATPase subunit isoforms in transepithelial transport.Key words: preimplantation development, trophectoderm, fluid transport, Na+/K+-ATPase, α subunit, β subunit, γ subunit.

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V. The epithelial sodium channel and its implication in human diseases

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1999.276.3.g567 ◽

1999 ◽

Vol 276 (3) ◽

pp. G567-G571 ◽

Cited By ~ 30

Author(s):

Edith Hummler ◽

Jean-Daniel Horisberger

Keyword(s):

Distal Colon ◽

Loss Of Function ◽

Newborn Mice ◽

Α Subunit ◽

Salt Wasting ◽

Lung Fluid ◽

Wasting Syndrome ◽

Rate Limiting Step ◽

Γ Subunit ◽

Rate Limiting

The epithelial Na+ channel (ENaC) controls the rate-limiting step in the process of transepithelial Na+ reabsorption in the distal nephron, the distal colon, and the airways. Hereditary salt-losing syndromes have been ascribed to loss of function mutations in the α-, β-, or γ-ENaC subunit genes, whereas gain of function mutations (located in the COOH terminus of the β- or γ-subunit) result in hypertension due to Na+ retention (Liddle’s syndrome). In mice, gene-targeting experiments have shown that, in addition to the kidney salt-wasting phenotype, ENaC was essential for lung fluid clearance in newborn mice. Disruption of the α-subunit resulted in a complete abolition of ENaC-mediated Na+ transport, whereas knockout of the β- or γ-subunit had only minor effects on fluid clearance in lung. Disruption of each of the three subunits resulted in a salt-wasting syndrome similar to that observed in humans.

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The Mitochondrial Genome Integrity Gene, MGI1, of Kluyveromyces lactis Encodes the β-Subunit of F1-ATPase

Genetics ◽

10.1093/genetics/144.4.1445 ◽

1996 ◽

Vol 144 (4) ◽

pp. 1445-1454 ◽

Cited By ~ 3

Author(s):

Xin Jie Chen ◽

G Desmond Clark-Walker

Keyword(s):

Mitochondrial Genome ◽

Kluyveromyces Lactis ◽

Three Dimensional ◽

Genome Integrity ◽

Dimensional Structure ◽

Deletion Mutants ◽

Β Subunit ◽

Gain Of Function ◽

F1 Atpase ◽

Γ Subunit

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the β-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the “gain-of-function” phenotype found in mgi1 point mutants. The location of Arg435 in the β-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the β- and α- (MGI2) subunits with the γ-subunit (MGI5) is likely to be affected by the mutations.

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Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

Endocrinology ◽

10.1210/en.2006-1499 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3977-3986 ◽

Cited By ~ 12

Author(s):

Satarupa Roy ◽

Sunita Setlur ◽

Rupali A. Gadkari ◽

H. N. Krishnamurthy ◽

Rajan R. Dighe

Keyword(s):

Chorionic Gonadotropin ◽

Human Chorionic Gonadotropin ◽

Expression System ◽

Biologically Active ◽

Chain Molecule ◽

Dependent Manner ◽

Β Subunit ◽

Single Chain ◽

Α Subunit ◽

Lh Receptor

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

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Thyroid hormone regulation of mRNAs encoding thyrotropin β-subunit, glycoprotein α-subunit, and thyroid hormone receptors α and β in brain, pituitary gland, liver, and gonads of an adult teleost, Pimephales promelas

Journal of Endocrinology ◽

10.1677/joe-08-0472 ◽

2009 ◽

Vol 202 (1) ◽

pp. 43-54 ◽

Cited By ~ 56

Author(s):

Sean C Lema ◽

Jon T Dickey ◽

Irvin R Schultz ◽

Penny Swanson

Keyword(s):

Thyroid Hormone ◽

Pituitary Gland ◽

Teleost Fish ◽

Hormone Receptors ◽

Pimephales Promelas ◽

Early Gene ◽

Thyroid Hormone Receptors ◽

Β Subunit ◽

Glycoprotein Hormone ◽

Α Subunit

Thyroid hormones (THs) regulate growth, morphological development, and migratory behaviors in teleost fish, yet little is known about the transcriptional dynamics of gene targets for THs in these taxa. Here, we characterized TH regulation of mRNAs encoding thyrotropin subunits and thyroid hormone receptors (TRs) in an adult teleost fish model, the fathead minnow (Pimephales promelas). Breeding pairs of adult minnows were fed diets containing 3,5,3′-triiodo-l-thyronine (T3) or the goitrogen methimazole for 10 days. In males and females, dietary intake of exogenous T3 elevated circulating total T3, while methimazole depressed plasma levels of total thyroxine (T4). In both sexes, this methimazole-induced reduction in T4 led to elevated mRNA abundance for thyrotropin β-subunit (tshβ) in the pituitary gland. Fish treated with T3 had elevated transcript levels for TR isoforms α and β (trα and trβ) in the liver and brain, but reduced levels of brain mRNA for the immediate-early gene basic transcription factor-binding protein (bteb). In the ovary and testis, exogenous T3 elevated gene transcripts for tshβ, glycoprotein hormone α-subunit (gphα), and trβ, while not affecting trα levels. Taken together, these results demonstrate negative feedback of T4 on pituitary tshβ, identify trα and trβ as T3-autoinduced genes in the brain and liver, and provide new evidence that tshβ, gphα, and trβ are THs regulated in the gonad of teleosts. Adult teleost models are increasingly used to evaluate the endocrine-disrupting effects of chemical contaminants, and our results provide a systemic assessment of TH-responsive genes during that life stage.

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Neural regulation of the formation of skeletal muscle phosphorylase kinase holoenzyme in adult and developing rat muscle

Biochemical Journal ◽

10.1042/bj3250793 ◽

1997 ◽

Vol 325 (3) ◽

pp. 793-800 ◽

Cited By ~ 2

Author(s):

Dean C. NG ◽

Richard C. CARLSEN ◽

Donal A. WALSH

Keyword(s):

Skeletal Muscle ◽

Muscle Development ◽

Rapid Decline ◽

Phosphorylase Kinase ◽

Β Subunit ◽

Subunit Protein ◽

Subunit Mrna ◽

Γ Subunit ◽

Muscle Phosphorylase ◽

Denervated Muscles

Neural influences on the co-ordination of expression of the multiple subunits of skeletal muscle phosphorylase kinase and their assembly to form the holoenzyme complex, α4β4γ4δ4, have been examined during denervation and re-innervation of adult skeletal muscle and during neonatal muscle development. Denervation of the tibialis anterior and extensor digitorum longus muscles of the rat hindlimb was associated with a rapid decline in the mRNA for the γ subunit, and an abrupt decrease in γ-subunit protein. The levels of the α- and β-subunit proteins in the denervated muscles also declined rapidly, their time course of reduction being similar to that for the γ-subunit protein, but they did not decrease to the same extent. In contrast with the rapid decline in γ-subunit mRNA upon denervation, α- and β-subunit mRNAs stayed at control innervated levels for approx. 8–10 days, but then decreased rapidly. Their decline coincided very closely with the onset of re-innervation. Re-innervation of the denervated muscles, which occurs rapidly and uniformly after the sciatic nerve crush injury, produced an eventual slow and prolonged recovery of the mRNA for all three subunits and parallel increases in each of the subunit proteins. A similar co-ordinated increase of both subunit mRNA and subunit proteins of the phosphorylase kinase holoenzyme was observed during neonatal muscle development, during the period when the muscles were attaining their adult pattern of motor activity. The phosphorylase kinase holoenzyme remains in a non-activated form during all of these physiological changes, as is compatible with the presence of the full complement of the regulatory subunits. These data are consistent with a model whereby the transcriptional and translational expression of phosphorylase kinase γ subunit occurs only with concomitant expression of the α and β subunits. This would ensure that free and unregulated, activated γ subunit alone, which would give rise to unregulated glycogenolysis, is not produced. The data also suggest that control of phosphorylase kinase subunit expression and the formation of the holoenzyme in skeletal muscle is provided by the motor nerve, probably through imposed levels or patterns of muscle activity.

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Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

Biochemical Journal ◽

10.1042/bcj20161000 ◽

2017 ◽

Vol 474 (5) ◽

pp. 751-769 ◽

Cited By ~ 7

Author(s):

M. Kristian Koski ◽

Jothi Anantharajan ◽

Petri Kursula ◽

Prathusha Dhavala ◽

Abhinandan V. Murthy ◽

...

Keyword(s):

Three Dimensional ◽

Coiled Coil ◽

Β Subunit ◽

Dimerization Domain ◽

Α Subunit ◽

Protein Dimer ◽

Symmetric Molecule ◽

Asymmetric Shape ◽

Proline Rich Peptide ◽

Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

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Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane

International Journal of Molecular Sciences ◽

10.3390/ijms19113591 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3591 ◽

Cited By ~ 3

Author(s):

Aki Nishiyama ◽

Sakura Matsuta ◽

Genki Chaya ◽

Takafumi Itoh ◽

Kotaro Miura ◽

...

Keyword(s):

Plasma Membrane ◽

Higher Plants ◽

Rice Genome ◽

Amino Acid Residues ◽

Subunit Gene ◽

Α Subunit ◽

Γ Subunit ◽

Domain Antibody ◽

C Terminus ◽

Liquid Chromatography Tandem Mass

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and β subunits (Gβ), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

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