Biomedical subjects

D S Weiss

Publications and source records attributed to D S Weiss.

At least 55 records · Page 3Linked to original sources

PubMed1997

Two tyrosine residues on the alpha subunit are crucial for benzodiazepine binding and allosteric modulation of gamma-aminobutyric acidA receptors.

Benzodiazepines (BZs) exert their therapeutic effects in the mammalian central nervous system at least in part by modulating the activation of gamma-aminobutyric acid (GABA)-activated chloride channels. To gain further insight into the mechanism of action of BZs on GABA receptors, we have been investigating structural determinants required for the actions of the BZ diazepam (dzp) on recombinant alpha1 beta2 gamma2 GABA(A) receptors. Site-directed mutagenesis was used to introduce point mutations into the alpha1 and gamma2 GABA(A) receptor subunits. Wild-type and mutant GABA(A) receptors were then expressed in Xenopus laevis oocytes or human embryonic kidney 293 (HEK 293) cells and studied using two-electrode voltage-clamp and ligand-binding techniques. With this approach, we identified two tyrosine residues on the alpha1 subunit (Tyr159 and Tyr209) that when mutated to serine, dramatically impaired modulation by dzp. The Y209S substitution resulted in a >7-fold increase in the EC50 for dzp, and the Y159S substitution nearly abolished dzp-mediated potentiation. Both of these mutations abolished binding of the high affinity BZ receptor antagonist [3H]Ro 15-1788 to GABA(A) receptors expressed in HEK 293 cells. These tyrosine residues correspond to two tyrosines of the beta2 subunit (Tyr157 and Tyr205) previously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on the gamma2 subunit produced only a slight increase in the EC50 for dzp (approximately 2-fold) with no significant effect on the binding affinity of [3H]Ro 15-1788. These data suggest that Tyr159 and Tyr209 of the alpha1 subunit may be components of the BZ-binding site on alpha1 beta2 gamma2 GABA(A) receptors.

Allosteric Regulation↗

PubMed1997

Lactone modulation of the gamma-aminobutyric acid A receptor: evidence for a positive modulatory site.

The gamma-aminobutyric acid-A (GABA(A)) receptor complex is allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and also directly gate the channel. A variety of gamma-butyrolactone analogues also modulate GABA-induced currents, with some potentiating and others inhibiting. Because several gamma-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin-insensitive GABA(A) receptors were performed to analyze whether some gamma-thiobutyrolactones interact with two distinguishable sites on the GABA(A) receptor. beta-Ethyl-beta-methyl-gamma-thiobutyrolactone inhibited GABA-induced currents at low concentrations (0.001-1 mM), but potentiated GABA-induced currents at higher concentrations (3-10 mM) in wild-type alpha1beta2gamma2-subunit containing ionophores. The related alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone potentiated submaximal GABA currents in wild-type receptors at both low and high concentrations (0.1-10 mM). Mutations in the second transmembrane domain of alpha1, beta2, or gamma2 conferred picrotoxinin-insensitivity onto GABA(A) receptor complexes. When these mutated alpha1, beta2, or gamma2 subunits were incorporated into the receptor complex, beta-ethyl-beta-methyl-gamma-thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1-10 mM). Neither the potentiating activity nor the EC50 of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone changed in the mutant receptors. Further studies demonstrated that the mutations did not affect the EC50 of chlordiazepoxide or phenobarbital. These and our earlier results identify a modulatory site on the GABA(A) receptor distinct from that interacting with barbiturates, benzodiazepines, and steroids. Additionally, they show that the gamma-butyrolactones probably interact at two different sites on the ionophore to produce opposite effects on GABA-mediated current.

4-Butyrolactone↗

PubMed1997

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle.

ATP-Binding Cassette Transporters↗

PubMed1997

Posttraumatic stress disorder and functioning and quality of life outcomes in a nationally representative sample of male Vietnam veterans.

OBJECTIVE: Although posttraumatic stress disorder (PTSD) is a highly prevalent and often chronic condition, the relationship between PTSD and functioning and quality of life remains incompletely understood. METHOD: The authors undertook an archival analysis of data from the National Vietnam Veterans Readjustment Study. The study subjects consisted of the nationally representative sample of male Vietnam veterans who participated in the National Vietnam Veterans Readjustment Study. The authors estimated PTSD at the time of the interview with the Mississippi Scale for Combat-Related Posttraumatic Stress Disorder. They examined the following outcomes: diminished well-being, physical limitations, bed day in the past 2 weeks, compromised physical health status, currently not working, and perpetration of violence. Logistic models were used to determine the association between PTSD and outcome; adjustment was made for demographic characteristics and comorbid psychiatric and other medical conditions. RESULTS: The risks of poorer outcome were significantly higher in subjects with PTSD than in subjects without PTSD in five of the six domains. For the outcome domains of physical limitations, not working, compromised physical health, and diminished well-being, these significantly higher risks persisted even in the most conservative logistic models that removed the shared effects of comorbid psychiatric and other medical disorders. CONCLUSIONS: The suffering associated with combat related-PTSD extends beyond the signs and symptoms of the disorder to broader areas of functional and social morbidity. The significantly higher risk of impaired functioning and diminished quality of life uniquely attributable to PTSD suggests that PTSD may well be the core problem in this group of difficult to treat and multiply afflicted patients.

Combat Disorders↗

PubMed1997

Post-traumatic stress disorder and functioning and quality of life outcomes in female Vietnam veterans.

OBJECTIVE: This investigation assessed whether current post-traumatic stress disorder (PTSD) was associated with impaired functioning in a nationally representative sample of female Vietnam veterans. METHODS: Logistic models were used to determine the association between PTSD and outcome while adjusting for demographic characteristics and medical and psychiatric co-morbidities. RESULTS: PTSD was associated with significantly elevated odds of poorer functioning in five of the six outcome domains; only the association between perpetration of violence in the past year and PTSD did not achieve statistical significance. After adjusting for demographics and medical and psychiatric co-morbidities, PTSD remained associated with significantly elevated odds of bed days, poorer physical health, and currently not working. CONCLUSIONS: Among female Vietnam veterans PTSD is associated with a broad profile of functional impairment. The significantly increased odds of impaired functioning and diminished quality of life suggest that PTSD may be the core problem of the set of problems afflicting female Vietnam veterans.

Activities of Daily Living↗

PubMed1996

Stoichiometry of a recombinant GABAA receptor.

GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABAA receptor/pore complex is presumed to be a pentamer typically composed of a combination of alpha, beta, and gamma subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAA receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat alpha 1(alpha L263S), beta 2(alpha L259S), and gamma 2(alpha L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., alpha and alpha L263S), along with their wild-type counter-parts (e.g., beta and gamma), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (alpha, beta, and gamma) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant alpha 1 beta 2 gamma 2 GABAA receptor is a pentamer composed of two alpha subunits, two beta subunits, and one gamma subunit.

Amino Acid Sequence↗

PubMed1996

Repressor forms of the enhancer-binding protein NrtC: some fail in coupling ATP hydrolysis to open complex formation by sigma 54-holoenzyme.

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between sigma54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by sigma70-holoenzyme. In this paper we characterize "repressor" mutant forms of NtrC from Salmonella typhimurium, forms that have lost the ability to activate transcription by sigma54-holoenzyme (in vitro activity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by sigma70-holoenzyme. The amino acid substitutions in NtrCrepressor proteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressor proteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrC(G219K) protein has "improved" DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressor proteins have impaired ATPase activity. We now show that two other repressor proteins, NtrC(A216V) and NtrC(A220T), have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrC(constitutive) protein, which is capable of activating transcription. These results demonstrate that NtrC(A216V) and NtrC(A220T) fail in a function of the central domain other than ATPase activity. Although they may fail in contact with sigma54-holoenzyme per se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.

Adenosine Triphosphatases↗

PubMed1996

Insights into the activation mechanism of rho1 GABA receptors obtained by coexpression of wild type and activation-impaired subunits.

To gain insight into the activation mechanism of homomeric ligand-gated receptor-channels, we examined human homomeric rho1 GABA receptors with fewer than the normal number of agonist binding sites. This was accomplished by coexpressing different ratios of wild type and activation-impaired rho1 subunits. Dose-response relations from oocytes coexpressing wild type and mutant subunits were comprised of two components in terms of GABA sensitivity; one 'wild type'-like and the other 'mutant'-like. Applying the binomial hypothesis to subunit coassembly enabled use to correlate these two components of the GABA dose-response relations to the underlying chimaeric receptor subtypes. We demonstrate that the receptors activate near normal provided that they are comprised of at least three wild type subunits. Our data are consistent with five equivalent and independent GABA binding sites of which only three need bind GABA to open the pore. The two additional binding sites may increase the GABA sensitivity of the rho1 receptor and, when bound by agonist, stabilize the open state.

Animals↗

PubMed1996

Stress responses of emergency services personnel to the Loma Prieta earthquake Interstate 880 freeway collapse and control traumatic incidents.

A three-group quasi-experimental design contrasted the responses of rescue workers to the 1989 Loma Prieta earthquake Interstate 880 freeway collapse (n = 198) with responses to critical incident exposure of Bay Area Controls (n = 140) and San Diego Controls (n = 101). The three groups were strikingly similar with respect to demographics and years of emergency service. The I-880 group reported higher exposure, greater immediate threat appraisal, and more sick days. The three groups did not differ on current symptoms. For the sample as a whole EMT/Paramedics reported higher peritraumatic dissociation compared with Police. EMT/Paramedics and California road workers reported higher symptoms compared with Police and Fire personnel. Nine percent of the sample were characterized as having symptom levels typical of psychiatric outpatients. Compared with lower distress responders, those with greater distress reported greater exposure, greater peritraumatic emotional distress, greater peritraumatic dissociation, greater perceived threat, and less preparation for the critical incident.

Disasters↗

PubMed1996

The hemA gene encoding glutamyl-tRNA reductase from the archaeon Methanobacterium thermoautotrophicum strain Marburg.

In archaea the first general tetrapyrrole precursor 5-aminolevulinic acid (ALA) is formed via the tRNA-dependent five-carbon pathway from glutamate. We have cloned the hemA gene encoding the central enzyme of the pathway glutamyl-tRNA reductase from the methanogenic archaeon Methanobacterium thermoautotrophicum by complementation of an Escherichia coli hemA mutant to ALA prototrophy. An 1194 bp open reading frame that encodes a 398 amino acid polypeptide with the calculated M, 44,509 was detected. The deduced amino acid sequence showed 20-35% amino acid identity to bacterial HemAs with the highest identity score to the Pseudomonas aeruginosa HemA. An identity of approximately 22% was found to plant HemAs. Glutamyl-tRNA reductase activity was shown for the M. thermoautotrophicum HemA after overexpression in E. coli and partial purification. The enzymatic reaction catalyzed by the partially purified enzyme revealed a temperature optimum of 65 degrees C at an optimal pH of 7.0. The reductase utilized preferentially NADPH for the reduction of the activated carboxyl group. The presence of ATP and GTP showed no obvious influence on catalysis.

Aldehyde Oxidoreductases↗

PubMed1996

The relationship of peritraumatic dissociation and posttraumatic stress: findings in female Vietnam theater veterans.

This study examined the relationship of dissociation at the time of trauma, as assessed by the Peritraumatic Dissociation Experiences Questionnaire, Rater Version (PDEQ-RV; C.R. Marmar, D.S. Weiss, & T.J. Metzler, in press), and posttraumatic stress symptoms in a group of 77 female Vietnam theater veterans. PDEQ-RV ratings were found to be associated strongly with posttraumatic stress symptomatology, as measured by the Impact of Event Scale (M.J. Horowitz, N. Wilner, & W. Alvarez, 1979), and also positively associated with level of stress exposure and general dissociative tendencies, measured by the Dissociative Experiences Scale. The PDEQ-RV was unassociated with general psychiatric symptomatology, as assessed by the clinical scales of the Minnesota Multiphasic Personality Inventory-2 (J.N. Butcher, W.G. Dahlstrom, J.R. Graham, A. Tellegen, & B. Kaemmer, 1989). The PDEQ-RV was predictive of posttraumatic stress symptoms beyond the contributions of level of stress exposure and general dissociative tendencies. The findings provide further support for the reliability and validity of the PDEQ-RV as a measure of peritraumatic dissociation.

Adult↗

PubMed1996

Sustained nicotine exposure differentially affects alpha 3 beta 2 and alpha 4 beta 2 neuronal nicotinic receptors expressed in Xenopus oocytes.

To determine whether prolonged exposure to nicotine differentially affects alpha 3 beta 2 versus alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 microM nicotine for alpha 4 beta 2 and alpha 3 beta 2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of alpha 4 beta 2 receptors decreased in a concentration-dependent manner with IC50 values in the 10 nM range following incubation for 24 h and in the 1 nM range following incubation for 48 h. In contrast, responses of alpha 3 beta 2 receptors following incubation for 24-48 h with 1,000 nM nicotine decreased by only 50-60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for alpha 4 beta 2 and alpha 3 beta 2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for alpha 4 beta 2 and alpha 3 beta 2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but alpha 4 beta 2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.

Animals↗

PubMed1996

Characteristics of emergency services personnel related to peritraumatic dissociation during critical incident exposure.

OBJECTIVE: The aim of this study was to identify characteristics of emergency services personnel related to acute dissociative responses at the time of critical incident exposure, a phenomenon designated "peritraumatic dissociation." METHOD: The authors studied 157 rescue workers who responded to the Nimitz Freeway collapse during the 1989 Loma Prieta earthquake in the San Francisco Bay Area as well as 201 rescue workers who were not involved in that disaster. Demographics, level of critical incident exposure, perceived threat at the time of exposure, personality attributes (assessed by the Hogan Personality Inventory), coping strategies (assessed by the Ways of Coping Questionnaire), and locus of control were related to subjects' scores on the Peritraumatic Dissociative Experiences Questionnaire. RESULTS: According to univariate tests, the subjects with clinically meaningful levels of peritraumatic dissociation were younger; reported greater exposure to critical incident stress; felt greater perceived threat; had lower scores on the adjustment, identify, ambition, and prudence scales of the Hogan Personality Inventory; had higher scores on measures of coping by means of escape-avoidance, self-control, and active problem solving; and had greater externality in locus of control. Linear modeling with multiple logistic regression analyses indicated that greater feelings of perceived threat, coping by means of escape-avoidance, and coping by means of self-control were associated with a greater likelihood of being in the peritraumatic dissociation group, above and beyond age and exposure to stress. CONCLUSIONS: Rescue workers who are shy, inhibited, uncertain about their identity, or reluctant to take leadership roles, who have global cognitive styles, who believe their fate is determined by factors beyond their control, and who cope with critical incident trauma by emotional suppression and wishful thinking are at higher risk for acute dissociative responses to trauma and subsequent posttraumatic stress disorder.

Adaptation, Psychological↗

PubMed1996

Open trial of fluvoxamine treatment for combat-related posttraumatic stress disorder.

A 10-week open-label trial of fluvoxamine was conducted for male Vietnam combat veterans with chronic PTSD. Subjects were excluded if they met full current criteria for panic disorder or agoraphobia, and lifetime criteria for psychosis, bipolar disorder, or organic mental syndrome. Repeated MANOVA was performed to determine change over time. Fluvoxamine was well tolerated; side effects were observed primarily early in treatment with headache, insomnia, sedation, and gastrointestinal distress being most frequent. Fluvoxamine was effective for treating the core intrusion, avoidance, and arousal symptoms of PTSD. Large treatment effects were seen by 4-6 weeks, and maintained at 10 weeks. The magnitude of change was greater than has been previously reported for antidepressant treatment of male Vietnam combat veterans with PTSD.

Ambulatory Care↗

PubMed1996

Crewmember interactions during a Mir space station simulation.

BACKGROUND: Interpersonal problems may negatively affect crews on long-duration space missions. These problems stem from crewmember tension and its displacement to the outside monitoring personnel and from disruptions in crew cohesion and unclear leadership roles. HYPOTHESES: We hypothesized that crew tension and dysphoria would transiently increase following stressful events and be greater in the second half of a mission; that cohesion would be less during the second half of a mission; that tension and dysphoria would be displaced to the outside monitoring personnel; and that high levels of leader support and control would produce high levels of cohesion. METHODS: We tested these hypotheses during a 135-d Mir space station simulation study in Moscow. At weekly intervals, the three crewmembers completed items from two group climate questionnaires, a mood questionnaire, and a log of stressful events. RESULTS: Contrary to expectations, there was significantly (p < 0.05) more total mood disturbance and tension during the first 9 wks than during the subsequent 10 wks of the simulation. Although levels of cohesion remained the same over time, cohesion scores dropped at a significantly greater rate during the last third of the seclusion. There was evidence for the displacement of tension and dysphoria to the outside monitoring personnel. There were significant correlations in the predicted direction between leader support and control and crew cohesion, as well as evidence of status leveling in the mission commander. CONCLUSIONS: Crewmember tension, cohesion, and leadership are important issues affecting people working in secluded environments, and they need to be studied further in space.

Adult↗

PubMed1995

Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain.

Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme. To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer. In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated. The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation. The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria). NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription. Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms. Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain. This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding. Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.

Alleles↗

PubMed1995

Formylmethanofuran:tetrahydromethanopterin formyltransferase (Ftr) from the hyperthermophilic Methanopyrus kandleri. Cloning, sequencing and functional expression of the ftr gene and one-step purification of the enzyme overproduced in Escherichia coli.

Methanopyrus kandleri is a methanogenic Archaeon that grows on H2 and CO2 at a temperature optimum of 98 degrees C. The gene ftr encoding the formylmethanofuran:tetrahydromethanopterin formyltransferase, an enzyme involved in CO2 reduction to methane, has been cloned, sequenced, and overexpressed in Escherichia coli. The overproduced enzyme could be purified in yields above 90% by simply heating the cell extract to 90 degrees C in 1.5 M K2HPO4 pH 8.0 for 30 min. From 1 g wet cells (70 mg protein) approximately 14 mg formyltransferase was obtained. The purified enzyme showed essentially the same catalytic properties as that purified from M. kandleri cells. The primary structure and properties of the formyltransferase are compared with those of the enzyme from Methanobacterium thermoautotrophicum (growth temperature optimum 65 degrees C) and Methanothermus fervidus (83 degrees C). Of the three enzymes that from M. kandleri had the lowest isoelectric point (4.2) and the lowest hydrophobicity of the amino acid composition. The enzyme from M. kandleri had the relatively highest content in alanine, glutamate and glutamine and the relatively lowest content in isoleucine, leucine and lysine. These properties, some of which are unusual for enzymes from other hyperthermophilic organisms, may reflect that the formyltransferase from M. kandleri is adapted to both hyperthermophilic and halophilic conditions.

Amino Acid Sequence↗

PubMed1995

The energy conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum is composed of eight different subunits.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane-associated enzyme complex which catalyzes an energy-conserving, sodium-ion-translocating step in methanogenesis from H2 and CO2. We report here that the complex is composed of eight different subunits for which evidence was obtained at the protein, DNA and RNA levels: (a) SDS/PAGE of the purified complex revealed the presence of eight different polypeptides of apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB) and 12 kDa (MtrF). The N-terminal amino acid sequences of the 12-, 12.5- and 13-kDa polypeptides, which had previously not been accessible, were determined; (b) cloning and sequencing of the corresponding genes revealed the presence of the eight mtr genes organized in a 4.9-kbp gene cluster in the order mtrEDCBAFGH; (c) Northern-blot analysis revealed the presence of a 5-kbp transcript. DNA probes derived from the mtrE and mtrH genes hybridized to the transcript, indicating that the eight mtr genes are organized in a transcription unit. By primer extension, the 5' end of the mtrEDC-BAFGH mRNA was analyzed. The mtr operon was found to be located between the methyl-coenzyme M reductase I operon (mcr) and a downstream open reading frame predicted to encode a Na+/Ca2+, K+ exchanger.

Amino Acid Sequence↗