Bound nucleotides and phosphorylation in Rhodospirillum rubrum.
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Adenosine Triphosphatases↗
Biomedical subjects
D A Harris
Publications and source records attributed to D A Harris.
Different species of alpha motoneurons in same pool: further evidence from effects of inhibition on their firing rates.
1. Single units of the plantaris pool were isolated in ventral root filaments of decerebrate cats and their critical firing levels (CFLs) were determined. Motoneurons of similar size were compared in firing rate (FR) during repetitive stimulation of the plantaris nerve to establish control values and also during added stimulation of various inhibitory nerves (sural, hamstrings, or peroneal). 2. Criteria, based on maximal firing rate, were developed whereby certain pairs of units of similar size could be reliably classified into different types. 3. A second, independent set of criteria was formulated by which the same pair of units could be classified according to their responses to added inhibitory inputs. 4. The ability to distinguish motoneurons consistently by more than one set of criteria reinforces the evidence that different physiological types of units exist within a single motoneuron pool. 5. The findings indicate that different types of cells either receive different densities of input from certain inhibitory sources or that they react differentially to the same amounts of these inputs.
Action Potentials↗
Detecting non-valid hearing tests in industry.
As greater numbers of workers come under industrial hearing conservation programs, the problem of nonorganic hearing loss is become critical. The potential malingerer can be identified by behavioral observations both before and during the testing of hearing and by an analysis of test results. Manual, self-recording, and microprocessor audiometry all incorporate validity and reliability checks which assist in detecting the malingerer. Methods of documenting inaccurate hearing tests are detailed, along with ways of obtaining accurate follow-up tests. The key to alleviating the problem of malingering is early detection and immediate resolution of the problem.
Audiometry↗
Microprocessor versus self-recording audiometry in industry.
Auditory thresholds of 46 industrial workers tested on both a self-recording audiometer and a microprocessor audiometer showed significant differences at 2, 3, 6, and 8 kc/s with the self-recording instrument measuring lower thresholds by from 2.3 to 6.7 db. Differences appear to be the result of the psychophysical methods used and the use of pulsing versus steady tones. The differences, though significant, should be of little consequence in a carefully administered industrial hearing conservation program.
Adult↗
Microprocessor, self-recording and manual audiometry.
AC HTLs were measured on 12 normal-hearing young adults by (a) standardized manual, (b) self-recording (Bekesy), and (c) microprocessor audiometry stimulating standard manual procedure, to determine how threshold measurements would be affected by the 3 methods of testing when utilized in industrial hearing conservation programs for compliance with federal agency recommendations. Self-recording audiometry resulted in mean thresholds lower than standard manual audiometry by from 1-9 db; microprocessor audiometry resulted in thresholds higher than standard manual audiometry by from 0.2-4.4 db except at 4 kc/s where the microprocessor mean threshold was 3.8 db lower. Thus microprocessor audiometry more closely approximated audiometry by standard manual procedure. All 3 methods required 7-8 min per S for a 7-frequency audiogram on both ears.
Adult↗
A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein.
1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.
Adenosine Triphosphatases↗
Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase.
1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2'-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. The nucleotide specificities of 'coupled processes' nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
Adenosine Diphosphate↗
The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light.
1. Photophosphorylation was studied in spinach chloroplasts on illumination, from the dark state, with saturating short ("single turnover") flashes of light. 2. At rapid flash rates (100 Hz), phosphorylation began within the first five flashes. The ATPase inhibitor protein appeared to be displaced from its inhibitory site on the ATPase also within five flashes, as deduced from the flash-induced ATPase activity. 3. At slower flash rates, or if the rate of electron transfer were reduced with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), phosphorylation began only after a larger number (50--60) of flashes. The displacement of the ATPase inhibitior protein was similarly delayed. 4. Partial displacement of the inhibitor protein from its inhibitory site on the ATPase (by pretreatment with dithioerythritol) allowed phosphorylation to proceed without a perceptible lag, even in the presence of DCMU. It was concluded that the ATPase inhibitor protein must be displaced on the ATPase before phosphorylation can begin, and that this process is energy dependent. 5. During the flash regime used, release of inhibitor from its inhibitory site seemed to be governed largely by the membrane potential. The light-induced pH gradient seemed to have little effect under these conditions. Our results are not compatible with a direct conformational interaction between the electron transfer chain and the ATPase causing displacement of the inhibitor. 6. The maximal rate of photophosphorylation induced by less than 200 flashes was 0.12--0.15 mol ATP made/mol ATPase per flash. This rate seemed to be limited not be the supply of energy to the ATPase molecules, nor by the maximal turnover capacity of the ATP synthesising system, but by the number of ATPase molecules which were active in synthesis, i.e., which lacked the inhibitor protein. 7. The bound nucleotides of the coupling ATPase exchanged with added nucleotides during single turnover flashes. At high flash rates, exchange began within 5 flashes. The average amount of nucleotide exchanged per flash over 100 flashes was about one tenth the amount of ATP synthesised in each flash. 8. It is concluded that, during phosphorylation, a steady state level of active coupling ATPases is set up. The energy-dependent displacement of the inhibitor protein, and its (energy-independent) relaxation back on to the inhibitory site are the two opposing factors involved in this steady state.
Adenosine Triphosphatases↗
The interactions of coupling ATPases with nucleotides.
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Adenine Nucleotides↗
Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system.
1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.
Adenosine Diphosphate↗
Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system.
1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.
Adenosine Diphosphate↗
The adenosine triphosphatase-inhibitor content of bovine heart submitochondrial particles. Influence of the inhibitor on adenosine triphosphate-dependent reactions.
1. The activity of the ATPase (adenosine triphosphatase) of phosphorylating particles prepared by sonication of bovine heart mitochondria in the presence of MgCl2 and ATP is influenced by the isolation method for the mitochondria used in the preparation of particles. Type-I particles, made from mitochondria isolated in a medium lacking succinate, have a lower ATPase activity than to Type-II particles, which are prepared from mitochondria isolated in a medium containing succinate. 2. Centrifugation under appropriate energized conditions increases the ATPase activity of Type-I particles almost to that of the Type-II particles. The ATPase activity of Type-II particles was only slightly stimulated by this procedure. These data are interpreted as indicating a higher content of the ATPase-inhibitor protein in the Type-I particles. 3. A comparison was made of the ATP-driven enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and the exchange of the endogenous tightly bound nucleotides of the ATPase in Type-I and Type-II particles. The effect of exogenous inhibitor protein on both these reactions was also studied. 4. The time-scale on which the inhibitor protein can exchange between ATPase molecules is discussed.
Adenine Nucleotides↗
Nucleotide-binding sites on the coupling adenosine triphosphatase from ox heart mitochondria [proceedings].
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Adenosine Diphosphate↗
Interaction of mitochondrial adenosine triphosphatase with nucleotides and with glycerol [proceedings].
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Adenosine Triphosphatases↗
Identification of two species of alpha motoneurons in cat's plantaris pool.
1. Single units of the plantaris pool were isolated in ventral root filaments of decerebrate cats and their critical firing levels (CFLs) were determined. Motoneurons of similar size, as judged by their CFLs and other criteria, were compared in firing rate (FR) during repetitive stimulation of the plantaris nerve. 2. Such units either differed very little or quite widely, suggesting that they were sampled randomly from two populations, one firing rapidly, the other slowly. The relationship between the two rates remained approximately constant, regardless of the intensity or rate of input the units received, as long as both of them discharged rhythmically. 3. In single experiments 10-15 of the smallest units in the pool (all with CFLs in the 0-8% range) were isolated and compared. Statistical analyses and visual inspection of these small samples again suggested the existence of two species of motoneurons. 4. Statistical analyses also indicated that the FRs of units in single experiments were not sampled from any one of a variety of parametric, single-modal distributions. This suggests that the data were sampled from a distribution having more than one mode, indicating the existence of separate populations or species of motoneurons among the small units of the pool (0-8% range of CFL). 5. Pooling of the normalized data from different experiments revealed a bimodal histogram, reinforcing the conclusion that there are two species of small alpha motoneurons in the plantaris pool.
Animals↗
Tightly bound nucleotides of the energy-transducing ATPase of chloroplasts and their role in photophosphorylation.
1. Like other energy-transducing membranes, chloroplast membranes bear a coupling ATPase with especially tight binding sites for adenine nucleotides. Membranes washed several times still contain 2.5 nmol ATP and 1.3 nmol ADP bound per mg chlorophyll, which is equivalent to 1.9 ATP and 1.0 ADP per coupling ATPase. 2. In de-energized membranes, these nucleotides exchange to only a limited extent with added nucleotides. In membranes illuminated in the presence of pyocyanine, however, complete exchange of the bound nucleotides occurs rapidly, irrespective of whether ATP or ADP is present in the medium. 3. Pi can exchange into these nucleotided at both the beta and gamma positions when the membranes are energized in the presence of Mg-2+. Equilibrium with the beta and gamma groups of th ebound nucleotides is, however, not complete. 4. The inhibitors and uncouplers Dio-9, S13 and EDTA have different effects on the exchange of nucleotides, the exchange of inorganic phosphate and photophosphorylation. 5. The bound ATP level on the membrane is stable to a wide variety of conditions. The ADP level, however, drops to near zero under conditions of maximal activation of the emmbrane ATPase.
Adenosine Diphosphate↗
Nucleotide-binding properties of native and cold-treated mitochondrial ATPase.
1. The bound nucleotides of the beef-heart mitochondrial ATPase (F1) are lost during cold inactivation followed by (NH4)2SO4 precipitation. The release of tightly bound ATP parallels the loss of ATPase activity during this process. 2. During cold inactivation, the sedimentation coefficient (s20, w) of the ATPase first declines from 12.1 S to 9 S, then to 3.5 S. (NH4)2SO4 precipitation of the 9-S component also leads to dissociation into subunits with s20, w of 3.5 S. 3. The 9-S component still contains the bound nucleotides, which are removed when it dissociated into smaller subunits. 4. Reactivation of cold-inactivated ATPase by incubation at 30 degrees C is increased by the presence of 25% glycerol. ATP, however, does not have any clearcut effect on the degree of reactivation in the presence of glycerol. 5. ADP is an inhibitor of the reactivation, probably because it exchanges during reactivation for bound ATP giving rise to an inactive 12-S component. 6. The exchange of tightly bound nucleotides with added adenine nucleotides is more extensive and faster with cold-inactivated ATPase than with the native enzyme. During reactivation up to 1.6 moles of ATP and 1.0 mole ADP can exchange per mole enzyme. 7. Incubation with GTP, CTP or inorganic pyrophosphate induces an increased activity of the ATPase, which, however, soon declines in the presence of ATP. It also disappears on precipitation of GTP-treated enzyme with (NH4)2SO4.
Adenosine Triphosphatases↗