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A J Murphy

Publications and source records attributed to A J Murphy.

At least 73 records · Page 4Linked to original sources
PubMed1985

"Transactivation" control signals in the promoter of the herpesvirus thymidine kinase gene.

The herpes simplex virus thymidine kinase (TK) gene is transcriptionally activated in trans ("transactivated") by virus-encoded proteins during the infectious cycle. We show that TK plasmids introduced into a HeLa cell transient transcription assay system are also transactivated after infection with a TK- virus. Several aspects of this response are similar to regulation during the normal infectious cycle. Assay of TK promoter deletion and 5- to 10-base-pair substitution mutants in this system reveals that the transactivation response depends on the intactness of 109 base pairs of 5' gene flanking sequence. Differences between these results and analogous assays in the Xenopus oocyte system are discussed. A model for the putative binding of transactivator(s) to the promoter region is presented.

Animals↗
PubMed1984

Nd3+ and Co2+ binding to sarcoplasmic reticulum CaATPase. An estimation of the distance from the ATP binding site to the high-affinity calcium binding sites.

Nd3+ binding to sarcoplasmic reticulum (SR) was detected by inhibition of ATPase activity and directly by a fluorimetric assay. Both methods indicated that Nd3+ inhibited the ATPase activity by binding in the high-affinity Ca2+ binding sites. The stoichiometry of binding was about 11 nmol of Nd3+ bound per mg of SR proteins at pNd = 6.5. At higher [Nd3+], substantial nonspecific binding occurred. The association constant for Nd3+ binding to the high-affinity Ca2+ binding sites was estimated to be near 2 X 10(9) M-1. When the CaATPase was inactivated with fluorescein isothiocyanate (FITC), 5.3 nmol were bound per mg of SR protein. This fluorescent probe is known to bind in the ATP binding site. The stoichiometry of Nd3+ binding to FITC-labeled CaATPase was the same, within experimental error, as to the unlabeled CaATPase. Fluorescence energy transfer between FITC in the ATP site and Nd3+ in the Ca2+ sites was found to be very small. This donor-acceptor pair has a critical distance of 0.93 nm and the distance between the ATP site and the closest Ca2+ was estimated to be greater than 2.1 nm. Parallel measurements with FITC-labeled SR and Co2+, an acceptor with a critical distance 1.2 nm, suggested the ATP and Ca2+ binding sites are greater than 2.6 nm apart.

Animals↗
PubMed1984

Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography.

Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.

Animals↗
PubMed1984

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium.

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.

Adenosine Triphosphate↗
PubMed1984

Interdependence of H+, Ca2+, and Pi (or vanadate) sites in sarcoplasmic reticulum ATPase.

The phosphorylation of sarcoplasmic reticulum ATPase with Pi in the absence of Ca2+ was studied by equilibrium and kinetic experimentation. The combination of these measurements was then subjected to analysis without assumptions on the stoichiometry of the reactive sites. The analysis indicates that the species undergoing covalent interaction is the tertiary complex E X Pi X Mg formed by independent interaction of the two ligands with the enzyme. The binding constant of Pi or Mg2+ to either free or partially associated enzyme is approximately equal to 10(2) M-1, and no significant synergistic effect is produced by one ligand on the binding of the other; the equilibrium constant (Keq) for the covalent reaction E X Pi X Mg E-P X Mg is approximately equal to 16, with kphosph = 53 s-1, and khyd = 3-4 s-1 (25 degrees C, pH 6.0, no K+). The phosphorylation reaction of sarcoplasmic reticulum ATPase with Pi is highly H+ dependent. Such a pH dependence involves the affinity of enzyme for different ionization states of Pi, as well as protonation of two protein residues per enzyme unit in order to obtain optimal phosphorylation. The experimental data can then be fitted satisfactorily assuming pK values of 5.7 and 8.5 for the two residues in the nonphosphorylated enzyme (changing to 7.7 for one of the two residues, following phosphorylation) and values of 50.0 and 0.58 for the equilibrium constants of the H2(E X HPO4) in equilibrium with H(E-PO3) + H2O and H(E X HPO4) in equilibrium with E-PO3 + H2O reactions, respectively. In addition to the interdependence of H+ and phosphorylation sites, an interdependence of Ca2+ and phosphorylation sites is revealed by total inhibition of the Pi reaction when two high affinity calcium sites per enzyme unit are occupied by calcium. Conversely, occupancy of the phosphate site by vanadate (a stable transition state analogue of phosphate) inhibits high affinity calcium binding. The known binding competition between the two cations and their opposite effects on the phosphorylation reaction suggest that interdependence of phosphorylation site, H+ sites, and Ca2+ sites is a basic mechanistic feature of enzyme catalysis and cation transport.

Adenosine Triphosphatases↗
PubMed1984

Cell-specified expression of a selectable hybrid gene.

We have inserted into a retroviral vector the structural information encoding the selectable bacterial genes neo and gpt linked, respectively, to the promoters of the herpesvirus thymidine kinase gene and the rat preproinsulin II gene. We have used this recombinant retrovirus construction to transduce insulinoma cell lines that are positive or negative for insulin expression. Selection of the transductants for neo yielded resistant colonies from all of the cell lines, while selection for gpt yielded resistant transductants in relatively high frequency only from insulin-producing cells. The 5' end of the gpt transcripts maps to the authentic preproinsulin capping site of the construction.

Animals↗
PubMed1983

Alterations in the structure of the ribose moiety of ATP reduce its effectiveness as a substrate for the sarcoplasmic reticulum ATPase.

The substrate specificity of the calcium ATPase of isolated sarcoplasmic reticulum vesicles was examined using a series of ribose-modified ATP analogs. Steady state hydrolytic rate measurements using analogs modified at the 2'- and 3'-positions demonstrate that both hydroxyl groups contribute to substrate specificity and high catalytic activity. The 3'-hydroxyl is especially significant in this regard since all analogs modified at the 3'-position were slowly hydrolyzed, if at all, and they did not produce the activation at millimolar concentrations characteristically observed with ATP. In contrast, ATP analogs modified only at the 2'-position were more rapidly hydrolyzed (although at rates less than for ATP), and they did produce activation at millimolar concentrations. These results suggest that neither the catalytic (high affinity) nor the regulatory (low affinity) site of the CaATPase tolerates changes in the hydroxyl substituent at the 3'-position of ATP, whereas steady state rates associated with substrate binding at both types of sites are less affected by changes in the 2'-hydroxyl of ATP.

Adenosine Triphosphatases↗
PubMed1982

Detergent-solubilized sarcoplasmic reticulum ATPase. Hydrodynamic and catalytic properties.

Solubilization of the ATPase of sarcoplasmic reticulum vesicles with the nonionic detergent dodecyl non-aoxyethylene alcohol (C12E9) resulted in a large (about 5-fold) increase in its Ca2+ ATPase activity. Measurements using a calcium ionophore suggest this activation was the result of rendering the vesicles permeable to calcium. Complete activity is preserved at a detergent concentration range in which the detergent is complexed with the monomeric form of the ATPase, as measured by Sepharose 6B chromatography. Using a calibrated column, we found the C12E9 complex to have a Stokes radius of 55 A. As measured by time-resolved fluorescence anisotropy decay experiments, it had a rotational correlation coefficient of 214 ns, which is equivalent to a Stokes radius of 59 A. The axial ratio of the corresponding ellipsoid of revolution is calculated to be 5 to 6, indicating the complex is quite asymmetric. Like the vesicular form of the ATPase, the detergent-solubilized monomeric form bound with high affinity about 9 nmol of Ca2+/mg of protein. Also, like the vesicular enzyme, the solubilized form displayed a Ca2+ dependence of the activation of ATP hydrolysis which was cooperative (Hill coefficient 1.8). These results suggest that the calcium sites interact intramolecularly.

Animals↗
PubMed1981

Dimensions of construing in psychiatric patients.

Analysis of the perceptions of schizophrenics suggests that there are significant differences between their constructions of the social world and those of normal Ss. The development of heuristic classifications, such as "psychological" and "nonpsychological," has demonstrated some of the dimensions on which perceptual differences may exist. The results of this study (N = 170) suggest that simple heuristic classifications may obscure some of the complexity involved in the analysis of social perceptions. X

Humans↗
PubMed1981

A double blind trial of extra-amniotic oestriol and prostaglandin F2 alpha gels in cervical ripening.

Forty nulliparous patients with Bishop score of 3 or less were given 15 mg oestriol gel, 10 mg PGF2 alpha gel or gel alone via the extra-amniotic route. Both the oestriol and prostaglandin treated patients had a significant increase in Bishop score and a significant reduction in induction-delivery interval compared to controls. Oestriol gel had significantly less stimulatory effect on uterine activity than prostaglandin gel, indicating a possible local action.

Cervix Uteri↗
PubMed1981

Fetal death following extra-amniotic prostaglandin gel. Report of two cases.

Intrauterine fetal death occurred in two pregnancies following the extra-amniotic insertion of prostaglandin gel to ripen the cervix. An antenatal cardiotocogram before gel insertion was normal in each case. Continuous external cardiotocography should be used in patients with a high risk pregnancy who undergo ripening of the cervix before induction of labour.

Adult↗
PubMed1981

"Spurious" labour - does it matter?

The obstetrical outcome of 357 pregnancies complicated by "spurious" labour was documented and compared with that in a control group. Patients with spurious labour were more likely to have had a spontaneous or induced abortion in the preceding pregnancy. In almost 85% of patients, the painful uterine contractions had ceased within 1 hour of hospital admission and there was no increased fetal morbidity in this group. There was a significantly increased incidence of meconium-stained liquor in patients who continued to have painful uterine contractions, but in whom labour did not ensue. Placental abnormalities were significantly more common in patients with spurious labour.

Amniotic Fluid↗
PubMed1980

Use of vaginal prostaglandin gel before induction of labour.

Tylose gel containing either 1.5 mg, 3.0 mg or 10.0 mg of prostaglandin F2 alpha was inserted into the posterior vaginal fornix of 165 patients on the evening before induction of labour. A control group of 100 patients received the gel alone. There was a significant reduction in the induction-delivery interval in nulliparae receiving at least 3.0 mg of prostaglandin, whereas, in multiparae all doses achieved this effect. There was also a significant reduction in the incidence of forceps delivery in nulliparae who received 3.0 mg or more of the prostaglandin gel; however, there was no difference in the incidence of spontneous labour, epidural anaesthesia or Caesarean section between the patients who received prostaglandin or those receiving gel alone.

Female↗