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D A Harris

Publications and source records attributed to D A Harris.

At least 19 recordsLinked to original sources
PubMed1992

Isolation and characterisation of a functional alpha beta heterodimer from the ATP synthase of Rhodospirillum rubrum.

An alpha beta heterodimer of the F1-ATPase of Rhodospirillum rubrum was isolated by extraction of chromatophores with LiCl. Each alpha beta heterodimer contains one tightly bound ADP, which is released upon removal of medium Mg2+. The dimer can be reversibly dissociated by removal of Mg(2+)-ions. The alpha beta heterodimer restores both ATP-synthetic and -hydrolytic activities to LiCl-treated chromatophores, saturation being achieved at approximately 2 mmol alpha beta.mol BChl-1. The heterodimer itself hydrolyses Mg-ATP with an activity distinct from RF1, being unaffected by azide or sulphite ions. The Vmax and Km (ATP) for this Mg(2+)-dependent activity were 110 +/- 10 nmol.min-1.mg protein-1 and 100 +/- 30 microM, respectively. The Km did not differ significantly from that of RF1.

Amino Acid Sequence↗
PubMed1992

Mitochondrial ATP synthase regulation in heart: defects in hypertension are restored after treatment with captopril.

Control of mitochondrial ATP synthase capacity was investigated in cultured cardiomyocytes from normotensive (Wistar-Kyoto) and spontaneously hypertensive rats. Cells from spontaneously hypertensive rats have a higher basal ATP synthase capacity than those from normotensives, but lack the normal up-regulation in response to an increased energy demand. After treatment of spontaneously hypertensive rats with captopril (60 mg/kg per day for 12 weeks), cellular hypertrophy characteristic of the hypertensives was abolished and the cardiomyocytes showed a normal ATP synthase capacity. Normal up-regulation of this enzyme was also restored. All cells showed a normal down-regulation of the synthase in response to cyanide. Experiments with the calcium antagonists, verapamil and ruthenium red, suggest that abnormal ATP synthase regulation observed in the untreated spontaneously hypertensive rats results from an alteration of Ca2+ handling in cardiac cells under chronic high workload, which is reversed by captopril treatment.

Animals↗
PubMed1991

A prion-like protein from chicken brain copurifies with an acetylcholine receptor-inducing activity.

The mammalian prion protein (PrPC) is a cellular protein of unknown function, an altered isoform of which (PrPSc) is a component of the infectious particle (prion) thought to be responsible for spongiform encephalopathies in humans and animals. We report here the isolation of a cDNA that encodes a chicken protein that is homologous to PrPC. This chicken prion-like protein (ch-PrLP) is identical to the mouse PrP at 33% of its amino acid positions, including an uninterrupted stretch of 24 identical residues, and it displays the same structural domains. In addition, ch-PrLP, like its mammalian counterpart, is attached to the cell surface by a glycosyl-phosphatidylinositol anchor. We find that ch-PrLP is the major protein in preparations of an acetylcholine receptor-inducing activity that has been purified greater than 10(6)-fold from brain on the basis of its ability to stimulate synthesis of nicotinic receptors by cultured myotubes. The ch-PrLP gene is expressed in the spinal cord and brain as early as embryonic day 6; and in the spinal cord, the protein appears to be concentrated in motor neurons. Our results therefore raise the possibility that prion proteins serve normally to regulate the chemoreceptor number at the neuromuscular junction and perhaps in the central nervous system as well.

Amino Acid Sequence↗
PubMed1991

Control of mitochondrial ATP synthase in rat cardiomyocytes: effects of thyroid hormone.

Activities of the mitochondrial ATP synthase and the electron transfer chain were investigated in cultured cardiomyocytes prepared from untreated and thyroxine-treated rats. Quiescent cells from the thyroxine-treated animals showed a 33% increase in mitochondrial ATP synthase capacity, but no change in respiratory chain capacity, relative to those from control animals. This increase was attributable largely to (a) a 25% increase in F1 content in these mitochondria, and partly to (b) a 10% stimulation in ATPase activity due to raised intramitochondrial Ca2+. Both types of cell showed a normal ATP content of 38-40 nmol/mg cell protein. In control cells, the mitochondrial ATP synthase responded to increased energy demand (by electrical stimulation and/or by positive inotropic agents) with an increase in its capacity of up to 2-fold. This response was absent in cells from thyroxine-treated animals. In addition, cellular ATP levels fell significantly after 2 min electrical stimulation of cells from thyroxine-treated animals, while those of control cells were constant. It was concluded that regulation of the mitochondrial ATP synthase was defective in heart cells from thyroxine treated rats, leading to an energy deficit when energy demand on the cells was increased. Animals treated with thyroxine, but allowed to recover for 17 days after treatment, showed responses indistinguishable from the control cells. Thus, the effects of thyroxine on mitochondrial activities were reversible.

Adenosine Triphosphate↗
PubMed1991

Cloning of non-polyadenylated RNAs from rat brain.

Rodent brain has been reported to contain a fraction of non-polyadenylated (poly(A)-) mRNA that includes about 100,000 different sequences, most of which are not found in the poly(A)+ fraction. We have prepared a cDNA library of low-abundance poly(A)- RNAs from rat brain polysomes, and have characterized three clones in detail. Two of the clones hybridize on Northern blots to poly(A)+ RNAs from brain. Dot blot hybridization and RNase protection assays demonstrate that although the bulk of the RNA complementary to these clones is present in the poly(A)- fraction, a small portion (7-21%) is present in the poly(A)+ fraction. Our results suggest that the poly(A)-mRNA fraction from rat brain may not contain sequences that are different from those in the poly(A)+ fraction.

Animals↗
PubMed1990

Promotion and inhibition of catalytic cooperativity of the Ca2+-dependent ATPase activity of spinach chloroplast coupling factor 1 (CF1).

ATP- and ITP-stimulation of the Ca2+-dependent hydrolysis of low concentrations of [gamma-32P]ATP was used as a direct demonstration of catalytic cooperativity in CF1. CF1 activated by epsilon-subunit removal or dithiothreitol, or by the presence of ethanol in the ATPase assay medium, shows pronounced catalytic cooperativity, with maximal stimulation of [gamma-32P]ATP hydrolysis at about 20 microM CaATP. Catalytic cooperativity is diminished by the presence of the epsilon-subunit or by pretreatment of either untreated or epsilon-depleted CF1 with azide (C1/2=30 microM). Both activated and untreated forms of CF1 also exhibit hydrolysis of CaATP by a high-affinity, low-capacity mode of turnover, which is unaffected by any of the preceding treatments and shows normal Michaelis-Menten behaviour. We propose that this high-affinity mode represents unisite catalysis, and that the endogenous inhibitor, epsilon, and the exogenous inhibitor, azide, both act exclusively on cooperative interactions between the catalytic sites.

Adenosine Triphosphate↗
PubMed1990

Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes.

The ATP synthase capacity of rat heart myocytes can be measured in sonicated cell suspensions and in sonicated preparations of cultured cardiomyocytes. This procedure allows the rapid measurement of mitochondrial function in response to changes in the metabolic status of the cell. In cultured myocytes, transitions in ATP synthase capacity (with no detectable change in cellular ATP concentration) accompany a change to anoxia or electrically stimulated contraction (rise of 70%). These changes are reversed on returning to the original conditions. Exposure of myocytes to low pH has little effect on basal ATP synthase capacity (down to values less than pH 6), but markedly affects cellular ATP levels and the response of the cells to anoxia and reoxygenation, possibly mimicking changes seen in ischaemic heart. Similar effects are seen in suspensions of freshly prepared myocytes, but these preparations are less stable and more pH-sensitive than are cells in culture. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase, and that a regulator protein, the naturally occurring inhibitor protein from mitochondria, may be responsible for this regulation.

Adenosine Triphosphate↗
PubMed1990

Control of mitochondrial ATP synthase in heart cells: inactive to active transitions caused by beating or positive inotropic agents.

STUDY OBJECTIVE - The aim of the study was to measure variations in ATP synthase capacity in cultured cardiomyocytes under conditions of metabolic stimulation. DESIGN - ATP synthase activity was measured in cultured rat cardiomyocytes using a procedure which allowed rapid measurement of mitochondrial function during changes in metabolic state. EXPERIMENTAL MATERIAL - Calcium tolerant cardiomyocytes were prepared from male Wistar rats, weight 250-300 g, n = 6-22 per experiment. MEASUREMENTS AND MAIN RESULTS - Electrical stimulation of cardiomyocytes led to an approximate doubling of ATP synthase capacity within 1-2 min, and was rapidly reversible. Activation was reduced when extracellular calcium was lowered and abolished in presence of the calcium entry blocker ruthenium red. Exposure of cardiomyocytes to isoprenaline or to an inhibitor of phosphodiesterase III also led to a large increase in ATP synthase capacity, which was abolished in presence of ruthenium red. However, the response of cells to isoprenaline depended on their pretreatment: activation of ATP synthase was abolished after 20 min anoxia prior to isoprenaline treatment but regained after a subsequent 30 min reoxygenation. This may reflect down regulation of beta receptors on the cell surface during anoxia. CONCLUSIONS - ATP synthase is directly controlled in vivo by a non-allosteric mechanism. Activation of ATP synthase is a response to intramitochondrial Ca2+ concentration.

Animals↗
PubMed1990

Defects in regulation of mitochondrial ATP synthase in cardiomyocytes from spontaneously hypertensive rats.

Control of mitochondrial ATP synthase capacity was investigated in cultured cardiomyocytes from normotensive (Wistar-Kyoto) and spontaneously hypertensive (SHR) rats. The basal ATP synthase capacity in quiescent cardiomyocytes was raised in the hypertensive rats (2.9 +/- 0.1 mumol.min-1.mg protein-1) compared with normotensive rats (2.2 +/- 0.1 mumol.min-1.mg-1). However, this high value is restored to normal after treatment of the cells with verapamil or ruthenium red; agents that do not affect ATP synthase capacity in normal cells. It is concluded that abnormally high intramitochondrial Ca2+ levels, or an abnormal mitochondrial response to Ca2+ concentration, are responsible for ATP synthase activation in quiescent SHR cells. Cardiomyocytes from normotensive rats respond to increased energy demand (caused by electrical stimulation or treatment with positive inotropic agents) by increasing their ATP synthase capacity up to twofold. Cells from SHR rats are unable to control their ATP synthase in this way. It is concluded that some defect in regulation of the mitochondrial ATP synthase occurs in myocytes from hypertensive rats.

Adenosine Triphosphate↗
PubMed1989

Reversible modulation of the mitochondrial ATP synthase with energy demand in cultured rat cardiomyocytes.

The ATP synthase capacity of rat heart myocytes can be measured in sonicates of cultured cardiomyocytes. In these cells, transitions in ATP synthase capacity occur on changing to the anoxic or uncoupled state (drop in ATP synthase capacity of over 40%) or on electrically stimulating the cells to contract (rise of 70%). These changes occur rapidly (half time less than 1 min) and are completely reversed on returning to the original conditions. It is proposed that mitochondria in vivo are directly regulated at the level of the ATP synthase. The naturally occurring inhibitor protein from mitochondria may be responsible for this regulation.

Adenosine Triphosphatases↗
PubMed1989

Azide as a probe of co-operative interactions in the mitochondrial F1-ATPase.

(1) The hydrolytic activity of the isolated mitochondrial ATPase (F1) is strongly inhibited by azide. However, at very low ATP concentration (1 microM or less), no inhibition by azide is observed. (2) The azide-insensitive ATPase activity represents a high-affinity, low-capacity mode of turnover of F1. This is identified with the low Km, low Vmax component seen in steady-state kinetic studies in the absence of azide. (3) The azide-insensitive ATPase activity shows simple Michaelis-Menten kinetics, with Km = 3.2 microM, and Vmax = 1.1 mumol/min per mg (6 s-1). It is unaffected by anions such as sulphite, or by increasing pH in the range 7 to 8, both of which stimulate the maximal activity of F1. (4) Both the azide-insensitive and azide-sensitive components of F1-ATPase activity are equally inhibited by labelling the enzyme with 7-chloro-4-nitrobenzofurazan, by binding the natural inhibitor protein, or by cold denaturation of the enzyme. (5) It is concluded that azide-insensitive ATP hydrolysis represents catalysis by F1 involving a single catalytic site, and that azide acts by abolishing intersubunit cooperativity between the three catalytic sites of F1. Azide-sensitivity is thus a useful probe for events which affect the active site of F1 directly.

Adenosine Triphosphate↗
PubMed1989

Differential activation of myotube nuclei following exposure to an acetylcholine receptor-inducing factor.

A glycoprotein purified from chick brain, of relative molecular mass 42,000, increases the rate of appearance of acetylcholine receptors (AChRs) on the surface of chick myotubes. RNase protection assays have shown that this AChR-inducing activity (ARIA) increases the amount of mRNA encoding the alpha-subunit of the AChR, with little or no effect on the amounts of gamma- and delta-mRNAs2. Here, we report that the mRNAs encoding the alpha- and gamma-subunits of the receptor detected by in situ hybridization are concentrated around nuclei in cultured myotubes. Consistent with previous results, ARIA selectively increased the amount of alpha-subunit mRNA, but we now find that all nuclei were not activated to the same extent, with a substantial number not responding at all. Assuming that ARIA is released by motor nerve terminals, our results indicate that only a subset of muscle nuclei are capable of contributing to the accumulation of AChRs at developing neuromuscular junctions.

Actins↗
PubMed1988

Kinetics of the release of the mitochondrial inhibitor protein. Correlation with synthesis and hydrolysis of ATP.

(1) The kinetics of the release of the mitochondrial inhibitor protein (IF1) is studied in bovine heart submitochondrial vesicles supplemented with 125I-labelled IF1, using a method for rapidly 'freezing' the state of F1-IF1 interaction. It is shown that generation of a protonmotive force leads to release of IF1 from F1 into solution, following an exponential process. (2) In one set of experiments the rate of IF1 release, in IF1 supplemented vesicles generating a protonmotive force, is correlated with the induction of ATP hydrolytic capacity. It is found that, even under different metabolic states (phosphorylating and non-phosphorylating conditions), both processes follow the same time-course (half-time of around 40 s) and that there is a direct correlation between induced ATPase capacity and IF1 released. This finding rules out the possibility of a non-inhibitory binding site for IF1 on the membrane. (3) In a second set of experiments, also using IF1 supplemented vesicles, the induction of the ATP hydrolytic capacity after energisation is correlated with the induction of the ATP synthetic capacity. Initial rates of both processes are monitored using firefly luciferase, keeping the assay systems as similar as possible. It is shown that the induction of each capacity follows an exponential time-course, with a half-time of around 40 s. This is in good agreement with the half-times obtained for the induction of ATP hydrolytic capacity and the rate of IF1 release, using the quench-stop method. (4) If the induction of ATP hydrolytic and synthetic capacities is followed in untreated submitochondrial vesicles, i.e., vesicles not supplemented with IF1, the extent and time-course of the change in both hydrolytic and synthetic capacities remain correlated, but the half-time of the transient falls to around 10 s. It is suggested that the length of the transient, observed in IF1 supplemented vesicles, results from partial loss of coupling during repeated centrifugations. (5) These results demonstrate that energy-dependent release of IF1 from F1 into solution results in a concomitant increase in both ATP synthetic and hydrolytic capacities of the ATP synthase complex, and that the time-course of this process is sensitive to the degree of coupling of the vesicles.

Adenosine Triphosphate↗
PubMed1988

The binding and release of the inhibitor protein are governed independently by ATP and membrane potential in ox-heart submitochondrial vesicles.

(1) The effects of membrane potential (delta psi) and nucleotides on the interaction between the F1-ATP synthase and its natural inhibitor protein (IF1) are studied in ox-heart submitochondrial vesicles. (2) Membrane potential causes displacement of IF1 from submitochondrial vesicles, as shown by measuring both delta psi-dependent stimulation of ATPase capacity and release of 125I-labelled IF1 from the vesicles. These effects are abolished if ATP is included in the incubation. (3) There is a linear increase in the steady-state ATPase capacity of oxidising vesicles as delta psi is increased from 100 mV to 135 mV. Increasing delta psi above 140 mV leads to no further change. (4) At a constant membrane potential, ATP suppresses the increase in ATPase capacity, with a concentration for half maximal effect of 140 microM. This value is close to the Km for ATP hydrolysis by membrane-bound F1. This suppression is related to ATP concentration rather than to delta Gp or ATP/ADP ratio. (5) The unidirectional on- and off-rates of IF1 were measured separately. The off-rate of IF1 is increased by membrane potential but unaffected by ATP. The on-rate, conversely, is increased by ATP. Thus, the suppression of the potential-dependent net release of IF1 from submitochondrial vesicles by ATP results from an increase of the IF1 on-rate above the off-rate.

Adenosine Triphosphate↗