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J A Cox

Publications and source records attributed to J A Cox.

At least 145 records · Page 8Linked to original sources
PubMed1982

Activation of human erythrocyte Ca2+-dependent Mg2+-activated ATPase by calmodulin and calcium: quantitative analysis.

The effect of Ca2+ and calmodulin on (CaM) on the activation of Ca2+-dependent Mg2+-activated ATPase (Ca2+,Mg2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) has been carried out because of the finding that the CaM dependence of the activation varies with the concentration of free Ca2+, similarly to brain phosphodiesterase and adenylate cyclase. The study was carried out in the absence of chelating agents because they strongly interfere in the enzyme kinetics. Three main conclusions can be drawn (i) CaM-Ca3 and CaM-Ca4 together are the biochemically active species in vitro. (ii) These species bind in a non-cooperative way to the CaM-binding site of the enzyme with a dissociation constant of 6 x 10(-10) M or 1.1 x 10(-8) M, depending on whether Ca2+ saturates the substrate binding site of the enzyme or not. (iii) The binding of CaM-Ca3 to the enzyme lowers the dissociation constant of the enzyme for Ca2+ at the substrate binding site from 51.5 to 2.8 microM. Contrary to general belief, CaM does not induce pronounced positive cooperativity in the binding of Ca2+ to the enzyme. Such a cooperativity is seen only when the enzyme is incompletely saturated with the activator, but it disappears in the presence of saturating concentrations of CaM-Ca3. The rate equation proposed here accurately predicts the extent of enzyme activation over a wide range of Ca2+ and CaM concentration. In healthy erythrocytes the concentrations of Ca2+ and CaM are such that the Ca pump works with a minimal dissipation of energy, but a small increase in the intracellular Ca2+ concentration leads to a strong amplification of the pumping activity.

Binding Sites↗
PubMed1982

Evidence for a role of calmodulin in the hydrosmotic action of vasopressin in toad bladder.

1. The informational role of cytosolic Ca2+ appears to be mediated by a ubiquitous protein--calmodulin--in most cell systems. 2. Evidence has been accumulating that not only cAMP, but also Ca2+, behaves as an intracellular messenger in the stimulation of water transport by vasopressin (hydrosmotic effect). 3. To examine whether calmodulin plays a role in the hydrosmotic effect of vasopressin, we used a specific antagonist of calmodulin--trifluoperazine--and looked at its effects on water transport in the urinary bladder of toads Bufo marinus. 4. The results showed that trifluoperazine, at micromolar concentrations, blocked the hydrosmotic effects of vasopressin or cAMP, thus indicating a post-cAMP site of action. 5. Two other psychotropic drugs--amitriptyline and harmaline--had similar effects, but higher concentrations were required to induce the same degree of inhibition of water flow. 6. Calmodulin was detected in the membrane and in the cytosolic fractions of isolated epithelial cells of toad bladder by means of the phosphodiesterase test. The content of both fractions was similar to that found in bovine brain. 7. The results suggest that calmodulin plays a regulatory role in the hydrosmotic action of vasopressin by possibly interacting with proteins associated with microfilaments and/or microtubules.

Amitriptyline↗
PubMed1981

Regulation of brain cyclic nucleotide phosphodiesterase by calmodulin. A quantitative analysis.

Comparison of the parameters of Ca and Sr binding to bovine brain calmodulin with the activation of bovine brain phosphodiesterase by Ca2+ and Sr2+ at different calmodulin concentrations allows a quantitative description of the mechanism of activation of the enzyme. Equilibrium dialysis studies show that calmodulin possesses three high affinity (K'diss = 6 micoM) and one low affinity (K'diss = 200 microM) sites for Ca2+. All four sites display the same affinity for Sr2+ with K'diss = 180 microM. In the presence of calmodulin, soluble bovine brain phosphodiesterase is activated by Sr2+ to the same extent as by Ca2+. The activation of the enzyme shows the same Ca2+/Sr2+ selectivity ratio of 30 as the binding of the metal ions to calmodulin. Based on the findings that the Ca2+ or Sr2+ concentration at half-maximal activation of the enzyme depends on the concentration of calmodulin present, a quantitative analysis of activation was carried out as a function of the four calmodulin-metal complex species (CaM . Men). The data show that the activating species are CaM . Ca3, CaM . Ca4 or CaM . Sr3, CaM . Sr4. The interaction of these activating species with phosphodiesterase follows the Hill equation with a dissociation constant of 10(-9) M and a Hill coefficient of 2, irrespective of the binding characteristics of Ca2+ or Sr2+. The latter value agrees well with the fact that phosphodiesterase possesses two binding sites for calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea.

A method is described for the rapid preparation of electrophoretically pure troponin C from rabbit skeletal-muscle myofibrils that avoids the use of urea. The three-step procedure includes extraction od the myofibrils with EDTA-containing buffers, one-step elution from DEAE-Sephadex and Sephadex G-100 chromatography in the presence of EDTA. The procedure gives yields comparable with those of currently used methods that involve dissociation of the troponin complex with urea. Except for the thiol-group reactivity, troponin C produced by our method is physicochemically and functionally indistinguishable from that obtained by the classical procedure. Purified troponin C always contains traces of calmodulin. However, this contamination can be decreased to less than 0.02% by means of a second Sephadex G-100 chromatography step in the presence of EDTA.

Animals↗
PubMed1980

Evidence for hemocyanin formation in the compound eye of Squilla mantis (Crustacea, Stomatopoda).

In the retina and in the subretinal space of the compound eye of Squilla mantis a special kind of pigment cell is present. The crystalline inclusions of this cell have been identified as hemocyanin, as determined (i) by the dimensional congruence of the crystalline substructure with the dimensions of isolated, purified hemocyanin, and (ii) by the immunofluorescence reaction using anti-hemicyanin antibodies. The ultrastructure of these cells, their location and the presence of crystalline bodies in their cytoplasm suggest that they are sites of hemocyanin synthesis and homologous to the cyanocytes or cyanoblasts of Limulus.

Animals↗
PubMed1980

A thermodynamic analysis of the binding of calcium and magnesium ions to parvalbumin.

Microcalorimetry and equilibrium dialysis were used to determine the thermodynamic functions delta H0, delta G0 and delta S0 guiding the interaction of Ca2+ and Mg2+ with purified carp muscle isoparvalbumin of pI 4.25. The equilibrium dialysis studies indicate equal affinities of the two metal sites of parvalbumin for either Ca2+ or Mg2+ with equilibrium constants of KCa = 2.7 X 10(9) M-1, and KMg = 9.5 X 10(4) M-1. Binding of the two metal ions is fully competitive with no indication for cooperative effects. The apparent Ca2+ affinity constant K'Ca in the presence of 1 mM Mg2+ is 2.8 X 10(7) M-1, and the Mg2+-Ca2+ exchange equilibrium constant equals 2.8 X 10(4) M-1. Microcalorimetry shows that parvalbumin exhibits negative reaction enthalpies of -37.2 kJ/mol metal site for Ca2+ binding and -25.1 kJ/mol site for Mg2+-Ca2+ exchange, yielding delta H0 = -12.1 kJ/mol site for Mg2+ complex formation. Enthalpy changes are linearly dependent upon the amount of metal bound to the protein, thus corroborating the equal affinities of the two sites. Reaction entropies delta S0 are +55.2 J x mol-1 x K-1 for Ca2+ complex formation and +54.8 x mol-1 x K-1 for Mg2+ complex formation. Thus the respective metal binding processes are driven by both enthalpy and entropy conbinations, and are reminiscent of Ca2+ binding to troponin C. The reaction entropy observed during Mg2+-Ca2+ exchange (0.4 J x mol-1 x k-1) is negligible in spite of the markedly different hydration entropies for Ca2+ and Mg2+. This indicates that the two metal complexes of parvalbumin do not have the same conformation entropy. Since no variation in the intrinsic protein fluorescence was observed upon metal exchange, the conformation differences must be restricted to the immediate environment of the metal binding sites.

Animals↗
PubMed1979

Renal homotransplantation in children.

Ninety-six renal transplants in 77 pediatric patients are reported with follow-up as long as 12 1/2 years. Thirteen of the first 14 patients are living with a functioning kidney after eight to 12 1/2 years. The patient survival for the entire group is 78%. Sixty-four percent are living with a functioning transplanted kidney. Splenectomy was initially performed at the time of transplant but has been discontinued because of concern that splenectomy in the immunosuppressed patient was related to an increased occurrence of septic complications. Anencephalic newborn infants have been found to be a satisfactory source of cadaver donor kidneys. Growth and development have been satisfactory when the transplant is performed prior to 12 years of age, if it functions well, and if an alternate-day regimen of steroid administration is followed. Both boys and girls have now passed through puberty with their transplanted kidneys, have married, become parents, and are leading essentially normal lives. A plea is made for earlier transplantation in small children with irreversible progressive renal failure before they develop severe stunting of growth and before the need for prolonged dialysis.

Adolescent↗
PubMed1979

Retroperitoneal lymph node dissection for Wilms' tumor.

Nephrectomy for Wilms' tumor was performed on 58 patients over a 20-yr period, and retroperitoneal lymphadenectomy was performed on 35 who presented with no demonstrable metastases. The survival rate was 100% for 19 clinical group I patients with negative nodes. Positive nodes in 9 instances led to 5 long-term survivors and significantly influenced staging as a guide for further therapy.

Humans↗
PubMed1979

Calcium, magnesium and the conformation of parvalbumin during muscular activity.

The conformation of perch parvalbumin in the Ca-, Mg- and metal-free state was studied by intrinsic fluorescence, trypsin susceptibility, thiol titration and circular dichroism. The data reveal that Ca-parvalbumin has a more compact structure than the metal-free protein, with a high alpha-helical content and a buried thiol. No difference in conformation could be detected between Mg- and Ca-parvalvumin, indicating that the Ca-Mg exchange that may take place during muscular activity is accompanied by little or no structural changes. Furthermore, recently published kinetic parameters can now be interpreted as meaning that, during the contraction-relaxation cycle, parvalbumin often stays in the Mg-form instead of switching to the Ca-form which is predominant in vitro.

Calcium↗