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Biomedical subjects

R Rodnight

Publications and source records attributed to R Rodnight.

At least 91 records · Page 5Linked to original sources
PubMed1974

Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of component of the sodium-plus-potassium ion-activited adenosine triphosphatase.

1. Ox brain microsomal fractions were labelled with [(32)P]ATP in the presence of Na(+) and the reaction was stopped with sodium dodecyl sulphate. The Na(+)-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [(32)P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na(+)-dependent label emerged in a peak corresponding to protein of molecular weight 570000-580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na(+)-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [(32)P]ATP in the absence of Na(+) revealed three labelled peaks, one of which corresponded in position to the Na(+)-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na(+)-dependent label. 4. Di-isopropyl [(32)P]phosphorofluoridate also labelled the band corresponding to the Na(+)-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50-60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.

Adenosine Triphosphatases↗
PubMed1974

Protein kinase activity stimulated by adenosine 3' :5'-cyclic monophosphate in synaptic-membrane fragments from ox brain. Inhibition of intrinsic activity by free and membrane-bound calcium ions.

1. Cyclic AMP-stimulated protein kinase activity phosphorylating intrinsic substrates in preparations of synaptic-membrane fragments from ox cerebral cortex was examined in relation to (a) the content of membrane-bound Ca(2+) in the preparations and (b) added Ca(2+) in the assay medium. 2. Centrifugal washing of synaptic-membrane fragments with buffered ethane dioxybis(ethylamine)tetra-acetate solutions decreased bound Ca(2+) from 2.8+/-0.4 (s.d.) to 0.9+/-0.3nmol/mg of protein. In washed preparations basal protein kinase activity was increased by about 40% and the cyclic AMP-stimulated activity by about 15%. Addition of Ca(2+) in the concentration range 5-50mum to the assay medium progressively inhibited the kinase activity of the washed preparations; in this range of Ca(2+) concentration the basal activity was inhibited more than the stimulated activity. 3. In unwashed preparations concentrations of Ca(2+) above 100mum inhibited the cyclic AMP-stimulated activity more than the basal activity. 4. The inhibitory effect of several concentrations of Ca(2+) was examined in relation to cyclic AMP concentration; no evidence for competition between Ca(2+) and cyclic AMP for a site on the enzyme was observed.

Animals↗
PubMed1973

Turnover of protein-bound serine phosphate in respiring slices of guinea-pig cerebral cortex. Effects of putative transmitters, tetrodotoxin and other agents.

1. The effect of various agents on the turnover of protein-bound phosphorus in respiring slices of cerebral cortex was studied. 2. Confirming previous work turnover was increased by the application of electrical pulses for 10s to the tissue. 3. Turnover was also increased by exposure of the slices for 10min to noradrenaline (0.5mm), 5-hydroxytryptamine (1mum) and histamine (0.1mm). 4. When slices were stimulated by electrical pulses in the presence of histamine the increase in turnover was the sum of the responses given by each agent above, suggesting that different phosphorylating systems were involved. 5. Tetrodotoxin (0.5mum) blocked the increased turnover due to electrical pulses, but not that due to histamine. Tetrodotoxin also prevented the increase in tissue cyclic AMP content caused by the application of electrical pulses. 6. Phosphoprotein turnover was not affected by adenosine, despite the increase in tissue cyclic AMP content given by this agent. 7. Adenosine blocked the phosphoprotein response to histamine, but did not affect the response to electrical pulses. 8. The results are discussed in relation to the hypothesis that the stimulation of protein phosphorus turnover by electrical pulses is secondary to the release of cyclic AMP in the tissue.

Adenosine↗
PubMed1973

Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3':5'-cyclic monophosphate.

1. Properties of the stimulation by cyclic AMP of the intrinsic protein kinase activity of membrane fragments from ox brain were studied. 2. Stimulation of activity declined from about 100% at 1min to less than 20% at 10min. The time-course was explained by the observation that cyclic AMP did not stimulate turnover of protein-bound serine phosphate once the membrane protein was fully phosphorylated. 3. Cyclic AMP accelerated the activity of a component of the basal activity rather than activating a different kinase. 4. The pH optimum for both the stimulated and basal activities was 7.2-7.4. NaCl (100mm) and KCl (10-100mm) inhibited the stimulated activity but did not affect the basal activity. 5. Strychnine and theophylline inhibited both activites equally, but the stimulated activity was more sensitive to inhibition by adenosine, bicuculline, vinblastin, veratrine, N-ethylmaleimide and cysteine. 6. No firm evidence for a role for endogenous cyclic AMP in the basal activity was found, but the possibility was not excluded. 7. Some 90% of both the stimulated and basal activities remained in an insoluble form after treatment of the membrane fragments with Triton X-100 (0.5%).

Adenosine↗
PubMed1973

The state of phosphorylation in vivo of membrane-bound phosphoproteins in rat brain.

The alkali-labile P content of membrane protein prepared from rapidly frozen rat brain was measured, CuSO(4) being used to inhibit protein phosphatase activity during subcellular fractionation. The P content of the membrane fraction was significantly increased (+12%) over the control value by incubation of homogenates with ATP before fractionation. This suggests that the membrane protein in rat brain is normally only partially phosphorylated.

Animals↗
PubMed1972

Determination of adenosine 3':5'-cyclic monophosphate in cerebral tissues by saturation analysis. Assessment of a method using a binding protein from ox muscle.

1. The Gilman (1970) procedure for determining cyclic AMP (adenosine 3':5'-cyclic monophosphate) by saturation analysis gave erroneous results when applied to the analysis of extracts of whole brain or preparations of membrane fragments from brain. 2. The extracts contained a non-diffusible factor, which enhanced the binding of cyclic AMP by the muscle protein fraction. 3. Extracts also contained material which inhibited binding, but net inhibition of binding was only observed when relatively concentrated extracts were analysed. 4. The error introduced by the factors modifying binding could be eliminated by incorporation of unlabelled internal standards in the unknowns. The design adopted enables a statistical estimate to be made of the standard error of a single assay. 5. The modified assay was used to determine bound cyclic AMP and adenylate cyclase activity in cerebral membrane fragments. Five preparations of synaptic membrane fragments contained less than 3.5pmol of cyclic AMP/mg of protein; a microsomal fraction from rat contained 65pmol of cyclic AMP/mg of protein.

Adenosine Triphosphate↗
PubMed1971

Protein-bound phosphorylserine in acid hydrolysates of brain tissue. The determination of ( 32 P)phosphorylserine by ion-exchange chromatography and electrophoresis.

1. Partial acid hydrolysates of proteins derived from cortical slices of guinea-pig brain were divided into two parts and fractionated by ion-exchange chromatography and high-voltage electrophoresis. 2. The apparent yield of protein-bound phosphorylserine by the ion-exchange method was about three times that obtained by electrophoresis. 3. The specific radioactivity of phosphorylserine isolated from (32)P-labelled slices by electrophoresis was twice that isolated by chromatography. 4. The discrepancies were found to be due to the presence of unlabelled phosphates of unknown composition in the ;phosphorylserine' fraction obtained by the ion-exchange method. 5. Electrical stimulation of slices respiring in the presence of [(32)P]phosphate increased the specific radioactivity of the total phosphate in the chromatographic ;phosphorylserine' fraction by 53+/-11%, as compared with only 19+/-5% for the phosphorylserine isolated by electrophoresis.

Animals↗
PubMed1971

Turnover of protein-bound phosphorylserine in membrane preparations from ox brain catalysed by intrinsic kinase and phosphatase activity.

1. The turnover of protein-bound phosphorylserine in preparations of membrane fragments from ox brain cortex was studied. 2. Turnover was considered to arise from the action of intrinsic protein kinases and phosphatases on a membrane protein or proteins. 3. Properties of the kinase system were studied by measuring the rate of incorporation of (32)P from gamma-labelled ATP into protein-bound phosphorylserine isolated from partial acid hydrolysates of membrane proteins. 4. Properties of the phosphatase system were studied by observing the rate of loss of (32)P from membrane preparations pre-labelled with [(32)P]ATP. 5. Net phosphorylation and dephosphorylation of membrane protein was observed during incubation of membrane preparations with and without ATP. 6. The rate of turnover was about 4nmol of P/h per mg of protein at 20 degrees C; dephosphorylation was considered to be the rate-limiting step.

Adenosine Triphosphate↗
PubMed1970

The effect of chemical agents on the turnover of the bound phosphate associated with the sodium-and-potassium ion-stimulated adenosine triphosphatase in ox brain microsomes.

1. The effect of chemical agents on the turnover of the Na(+)-dependent bound phosphate and the simultaneous Na(+)-dependent hydrolysis of ATP by a membrane preparation from ox brain was studied at an ATP/protein ratio of 12.5pmol/mug. 2. The agents were added immediately after phosphorylation of the preparation in a medium containing 50mm-sodium chloride and 2.5mum-[gamma-(32)P]ATP. 3. Concentrations of sodium chloride above 150mm, calcium chloride to 20mm and suramin to 1.4mm inhibited both phosphorylation and dephosphorylation and concomitantly slowed ATP hydrolysis. At 125mm-sodium chloride dephosphorylation and hydrolysis were slightly slowed without affecting phosphorylation. 4. Ethanol to 1.6m concentration inhibited dephosphorylation without affecting phosphorylation; the bound phosphate was increased and ATP hydrolysis slowed. 5. Ouabain to 4mm concentration partially inhibited ATP hydrolysis and caused a transient (1-2s) rise in bound phosphate followed by a rapid fall to a lower plateau value, which eventually declined to zero by the time ATP hydrolysis was complete. 6. Of the detergents examined Lubrol W, Triton X-100 and sodium deoxycholate had no significant effect on turnover. Sodium dodecyl sulphate and sodium decyl sulphate to 3.5mm and 20mm respectively completely inhibited turnover and ATP hydrolysis and stabilized the bound phosphate.

Adenosine Triphosphatases↗
PubMed1970

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex.

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.

Adenosine Triphosphatases↗