Biomedical subjects

D R Evans

Publications and source records attributed to D R Evans.

At least 55 records · Page 3Linked to original sources

PubMed1997

Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase.

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

Adenosine Triphosphate↗

PubMed1997

Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis.

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37 degrees), cells dependent solely on pph22 alleles for PP2A function displayed a rapid arrest of proliferation. Ts pph22 mutant cells underwent lysis at 37 degrees, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37 degrees. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24 degrees to 37 degrees, Ts- pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37 degrees, many Ts- pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts- pph22 mutant cells also accumulated aberrant microtubule structures at 37 degrees, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts- pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.

Actins↗

PubMed1997

The retirement adjustment process: changes in the well-being of male retirees across time.

The purpose of this prospective study was to (1) evaluate the impact of retirement, (2) monitor the change in adjustment across time, and (3) identify the resources predictive of short- and long-term adjustment in retirement. A sample of 117 male retirees was assessed on indices of physical and psychological health, perceived control, retirement satisfaction, and life satisfaction at 2-4 months preretirement, 1 year post-, and 6-7 years postretirement. The results provided support for a positive impact of retirement, as retirees evidenced increases in well-being during the first year. There was also evidence of a retirement adjustment process, in that aspects of well-being (i.e., psychological health) changed from short- to long-term retirement. Finally, physical health, income, and voluntary retirement status predicted short-term adjustment, while internal locus of control was an additional resource for long-term adjustment. Changes in resources over time also differentially predicted short- and long-term adjustment (e.g., an increase in internal locus of control predicted an increase in activity satisfaction at 1 year but not at 6-7 years postretirement).

Aged↗

PubMed1996

Function of the major synthetase subdomains of carbamyl-phosphate synthetase.

The amidotransferase domain (GLNase) of mammalian carbamyl-phosphate synthetase II hydrolyzes glutamine and transfers ammonia to the synthetase domain where carbamyl phosphate is formed in a three-step reaction sequence. The synthetase domain consists of two homologous subdomains, CPS.A and CPS.B. Recent studies suggest that CPS.A catalyzes the initial ATP dependent-activation of bicarbonate, whereas CPS.B uses a second ATP to form carbamyl phosphate. To establish the function of these substructural elements, we have cloned and expressed the mammalian protein and its subdomains in Escherichia coli. Recombinant CPSase (GLNase-CPS.A-CPS.B) was found to be fully functional. Two other proteins were made; the first consisted of only GLNase and CPS.A, whereas the second lacked CPS.A and had the GLNase domain fused directly to CPS.B. Remarkably, both proteins catalyzed the entire series of reactions involved in glutamine-dependent carbamyl phosphate synthesis. The stoichiometry, like that of the native enzyme, was 2 mol of ATP utilized per mol of carbamyl phosphate formed. GLN-CPS.B is allosterically regulated, whereas GLN-CPS.A was insensitive to effectors, a result consistent with evidence showing that allosteric effectors bind to CPS.B. These properties are not peculiar to the mammalian protein, because the separately cloned CPS.A subdomain of the E. coli enzyme was also found to catalyze carbamyl phosphate synthesis. Gel filtration chromatography and chemical cross-linking studies showed that these molecules are dimers, a structural organization that may be a prerequisite for the overall reaction. Thus, the homologous CPS.A and CPS.B subdomains are functionally equivalent, although in the native enzyme they may have different functions resulting from their juxtaposition relative to the other components in the complex.

Adenosine Triphosphate↗

PubMed1996

A study of letters between general dental practitioners and consultant orthodontists.

A survey was undertaken involving 268 general dental practitioners and 13 orthodontic consultants to determine the content of letters passed between them. There was good agreement between the two sets of respondents as to the appropriate contents of a letter. The feature of a letter least valued by both dentists and consultants was a detailed description of the case, although 54% of consultants described cases at some length when replying to referrers. The information that GDPs valued most highly was a treatment plan, the date on which the patient was seen and the name of the clinician involved, a short case description and the length of time before treatment would being if the patient was accepted for specialist treatment.

Communication↗

PubMed1996

Toward a general model of health-related quality of life.

A health-related quality of life (HRQOL) model that fitted data collected in previous studies from two separate samples--patients with chronic physical illness and patients with chronic mental illness--was adapted and tested on a representative sample drawn from the normal population. This model, which implied that both physical and mental health influenced number of friendships, personal growth, and job satisfaction, fitted the data very well. However, an alternative model reversing the directions of the pathways so that friendships, personal growth, and job satisfaction affected physical health and mental health, with the latter two having a mutual effect on each other, fitted the data even better. This suggests that, although a medical model of HRQOL may be more important when it comes to alleviating illness, a psychosocial model of HRQOL may be more important when it comes to maintaining health and preventing illness. Moreover, the fact that essentially the same five-factor structural model fits three distinct populations, healthy and unhealthy, suggests that there may be a general model of quality of life applicable to all populations. The implications of these findings for clinical theory, policy, and practice are discussed.

Adult↗

PubMed1995

Function of conserved histidine residues in mammalian dihydroorotase.

Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [14C]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Substructure of the amidotransferase domain of mammalian carbamyl phosphate synthetase.

The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of the 243-kDa CAD polypeptide, consists of a carboxyl half that is homologous to all trpG-type amidotransferases and an amino half unique to the carbamyl phosphate synthetases. The two halves of the mammalian GLNase domain have been cloned separately, expressed in Escherichia coli, and purified. The 21-kDa carboxyl half, the catalytic subdomain, is extraordinarily active. The kcat is 347-fold higher and the KGlnm is 40-fold lower than the complete GLNase domain. Unlike the GLNase domain, the catalytic subdomain does not form a stable hybrid complex with the E. coli CPSase synthetase subunit. Nevertheless, titration of the synthetase subunit with the catalytic subdomain partially restores glutamine-dependent CPSase activity. The 19-kDa amino half, the interaction subdomain, binds tightly to the E. coli CPSase large subunit. Thus, the GLNase domain consists of two subdomains which can autonomously fold and function. The catalytic subdomain weakly interacts with the synthetase domain and has all of the residues necessary for catalysis. The interaction subdomain is required for complex formation and also attenuates the intrinsically high activity of the catalytic subdomain and, thus, may be a key element of the interdomain functional linkage.

Animals↗

PubMed1995

Eukaryotic initiation factor 4E degradation during brain ischemia.

Suppression of protein synthesis in the brain following an ischemic insult has been thought to occur because of inhibition of translation initiation. All eukaryotic mRNAs, with the exception of heat-shock transcripts, require the activity of eukaryotic initiation factor (eIF) 4E for formation of the translation initiation complex, and eIF-4E availability is rate-limiting. The response of brain eIF-4E concentration and phosphorylation following decapitation ischemia was studied in rat brain homogenates after electrophoresis and western blotting with antibodies against eIF-4E and phosphoserine, respectively. There was no change in level of eIF-4E after 5 min of ischemia (p = 0.82 vs. time 0), but it had decreased 32 (p = 0.01) and 57% (p = 0.006) after 10 and 20 min of ischemia, respectively. There was no loss of serine phosphorylation on eIF-4E beyond signal loss observed due to degradation of the protein itself (p = 0.31). In vitro exposure of eIF-4E to activated mu-calpain resulted in a 50% loss in 10 min of eIF-4E on western blots. If active eIF-4E is required for translation of its own mRNA, degradation of this protein during ischemia, possibly by activated mu-calpain, could be a direct mechanism of irreversible neuronal injury, and the rate of proteolysis of eIF-4E could place an upper time limit on the maximal duration of global brain ischemia compatible with neurologic recovery.

Animals↗

PubMed1995

Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

Alleles↗

PubMed1994

Identification of the regulatory domain of the mammalian multifunctional protein CAD by the construction of an Escherichia coli hamster hybrid carbamyl-phosphate synthetase.

Carbamyl-phosphate synthetases from different organisms have similar catalytic mechanisms and amino acid sequences, but their structural organization, sub-unit structure, and mode of regulation can be very different. Escherichia coli carbamyl-phosphate synthetase (CPSase), a monofunctional protein consisting of amido-transferase and synthetase subunits, is allosterically inhibited by UMP and activated by NH3, IMP, and ornithine. In contrast, mammalian CPSase II, part of the large multifunctional polypeptide, CAD, is inhibited by UTP and activated by 5-phosphoribosyl-1-pyrophosphate (PRPP). Previous photoaffinity labeling studies of E. coli CPSase showed that allosteric effectors bind near the carboxyl-terminal end of the synthetase subunit. This region of the molecule may be a regulatory subdomain common to all CPSases. An E. coli mammalian hybrid CPSase gene has been constructed and expressed in E. coli. The hybrid consists of the E. coli CPSase synthetase catalytic subdomains, residues 1-900 of the 1073 residue polypeptide, fused to the amino-terminal end of the putative 190-residue regulatory subdomain of the mammalian protein. The hybrid CPSase had normal activity, but was no longer regulated by the prokaryotic allosteric effectors. Instead, the glutamine- and ammonia-dependent CPSase activities and both ATP-dependent partial reactions were activated by PRPP and inhibited by UTP, indicating that the binding sites of both of these ligands are located in a regulatory region at the carboxyl-terminal end of the CPSase domain of CAD. The apparent ligand dissociation constants and extent of inhibition by UTP are similar in the hybrid and the wild type mammalian protein, but PRPP binds 4-fold more weakly to the hybrid. The allosteric ligands affected the steady state kinetic parameters of the hybrid differently, suggesting that while the linkage between the catalytic and regulatory subdomains has been preserved, there may be qualitative differences in interdomain signal transmission. Nevertheless, switching prokaryotic and eukaryotic allosteric controls argues for remarkable conservation of structure and regulatory mechanisms in this family of proteins.

Adenosine Diphosphate↗

PubMed1994

Cloning and expression of the mammalian multifunctional protein CAD in Escherichia coli. Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker.

The multifunctional protein CAD catalyzes the first three steps in de novo pyrimidine biosynthesis in mammalian cells. Glutamine-dependent carbamyl-phosphate synthetase (CPSase), aspartate transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and aspartate transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in Escherichia coli. The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex mammalian protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of carbamyl phosphate, determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous carbamyl phosphate pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.

Animals↗

PubMed1994

Cloning, expression, and functional interactions of the amidotransferase domain of mammalian CAD carbamyl phosphate synthetase.

The trpG-type amidotransferases, a homologous but structurally diverse family of molecules, catalyze glutamine hydrolysis to supply ammonia for many biosynthetic reactions. The amidotransferase or glutaminase (GLNase) domain of mammalian carbamyl phosphate synthetase (CPSase), part of a 243-kDa polypeptide that initiates de novo pyrimidine biosynthesis, has been cloned and expressed in Escherichia coli. Complementation studies showed that a functional protein was produced in vivo which could provide ammonia for carbamyl phosphate synthesis by the host CPSase synthetase subunit. The recombinant 38-kDa protein was identified by immunoblotting, but when purified to homogeneity, had marginal glutaminase activity. Titration of the E. coli CPSase synthetase subunit with the mammalian GLNase domain resulted in the formation of a fully active 1:1 stoichiometric stable complex which catalyzed the glutamine-dependent overall reaction. The hybrid, isolated by gel filtration, had kinetic parameters (KGLNm = 102 microM, KATPm = 1.8 mM, kcat = 5.7 s-1) similar to those of the native E. coli CPSase. Thus, the amidotransferase activity of mammalian CPSase is carried by an autonomous domain which folds independently. However, optimal catalytic activity requires association of the glutaminase and synthetase domains. The conservation of this linkage in the mammalian E. coli hybrid suggests that the subunit interfaces must be nearly identical in the eukaryotic and prokaryotic proteins.

Animals↗

PubMed1994

Cell-cycle mutations among the collection of Saccharomyces cerevisiae dna mutants.

The temperature-sensitive dna mutants of the budding yeast Saccharomyces cerevisiae (Dumas et al. (1982) Mol. Gen. Genet. 187, 42-46) are more inhibited in DNA synthesis than in protein synthesis. These properties are also characteristic of many yeast mutations that inhibit progress through the cell cycle. Therefore we surveyed the collection of dna mutants for cell-cycle mutations. By genetic complementation we found that dna1 = cdc22, dna6 = cdc34, dna19 = cdc36, and dna39 = dbf3. Furthermore, by direct gene cloning we found that the dna26 mutation is allelic to prt1 mutations, which are known to exert primary inhibition on protein synthesis. This protein-synthesis mutation exerts a dna phenotype due to cell-cycle inhibition: prt1 mutations can block the regulatory step of the cell cycle while allowing significant amounts of protein synthesis to continue. Our non-exhaustive screening suggests that the dna mutants may house other mutations that affect the yeast cell cycle.

Alleles↗

PubMed1994