Direct and <sup>18</sup>O-Exchange Measurements Relevant to Possible Activated or Phosphorylated States of Myosin<sup>*</sup>

Sartorelli, L.; Fromm, Herbert J.; Benson, Robert W.; Boyer, Paul D.

doi:10.1021/bi00873a015

Cited by 55 publications

(23 citation statements)

References 28 publications

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“…[The process illustrated in Fig. 6(b) is unlikely to correspond to the rate of the reversal of the thioADP-Pi-heavy meromyosin complex to thioATP since this process is only very slowly reversible for ATP (Levy & Koshland, 1959;Sartorelli et al, 1966). ]…”

Section: Resultsmentioning

confidence: 99%

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Trentham

Bardsley

Eccleston

et al. 1972

Biochemical Journal

161

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Transient kinetic studies of Mg2+-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and Pi into the reaction medium by using linked assay systems. The release of Pi was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD+ in the presence of D-glyceraldehyde 3-phosphate, D-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and Pi, from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-f-D-ribofuranosylpurine 5'-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes ofthe mechanism. It was shown that the dissociation rate ofthioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3 s-1, which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.

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Section: Resultsmentioning

confidence: 99%

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Trentham

Bardsley

Eccleston

et al. 1972

Biochemical Journal

161

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“…The transition Y -_ X must therefore include the step of the cross-bridge cycle in which ATP is hydrolysed. The Pi so produced need not have been released from actomyosin, since the acid extraction step employed in the analysis removes nucleotides or Pi bound to actomyosin (Sartorelli, Fromm, Benson & Boyer, 1966).…”

Section: Discussionmentioning

confidence: 99%

High‐energy phosphate metabolism and energy liberation associated with rapid shortening in frog skeletal muscle

Homsher

Irving

Wallner

1981

The Journal of Physiology

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SUMMARY1. High-energy phosphate metabolism and energy liberated as heat and work were measured in 3 sec tetani of frog sartorius muscles at 0 OC.2. Three contraction periods were studied: (a) shortening at near-maximum velocity for 0-3 sec from sarcomere length 2-6 to 18 sum, beginning after 2 sec of isometric stimulation, (b) the 0 7 sec isometric period immediately following such rapid shortening, (c) the period from 2 to 3 sec in an isometric tetanus at sarcomere length 1P8 um.3. There were no significant changes in levels of ATP, ADP or AMP in any contraction period. The observed changes in inorganic phosphate and creatine levels indicated that the only significant reaction occurring was phosphocreatine splitting.4. The mean rate of high-energy phosphate splitting during rapid shortening, 0-48 + 0-24 ,imole/g . sec (mean + S.E. of mean, n = 29; 'g' refers to blotted muscle weight), was not significantly different from that in the 1 sec period in the isometric tetanus, 0-32 +011 smole/g. sec (n = 17). The mean rate in the post-shortening period, 0 71 +0 10 ,umole/g . sec (n = 22), was greater than that in the 1 sec period in the isometric tetanus, and this difference is significant (P < 0-02, t test).5. A large quantity of heat plus work was produced during the rapid shortening period, but less than half of this could be accounted for by simultaneous chemical reactions. The unexplained enthalpy production was 6-5 + 26 mJ/g (mean +S.E. of mean). No significant unexplained enthalpy was produced in the 1 sec period in the isometric tetanus.6. In the post-shortening period the observed enthalpy was less, by 6-2 + 2-6 mJ/g, than that expected from the simultaneous chemical reactions.7. The results are interpreted in terms of an exothermic shift in the population of cross-bridge states during rapid shortening. It is suggested that a relatively slow subsequent step prevents many of these cross-bridges from completing the cycle and splitting ATP until after the end of shortening.

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“…Fatty acid and succinic kinase activities of fractions obtained from rat kidney mitochondria Fractions derived from extracts of rat kidney mitochondria were assayed for fatty acid and succinic kinase activities by measuring the net decrease in CoA sulfhydryl [16]. Similar results were obtained for orthophosphate release [23] in GTP-succinate and GTP-fatty acid system. The fractions were obtained by suspending packed mitochondria (150mg of protein) in 5ml of 0.1 M Tris-buffer (pH 7.4).…”

Section: Resultsmentioning

confidence: 78%

Organization of Fatty Acid Oxidation in Rat Kidney Mitochondria

Róssi¹,

Alexandre²,

Sartorelli³

1968

European Journal of Biochemistry

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Long-chain fatty acids are oxidized by intact rat kidney mitochondria under experimental conditions which permit the location of three long-chain acyl-CoA synthesizing systems.Oleate oxidation which is 2,4-dinitrophenol-insensitive and which is inhibited by arsenate and excess phosphate, is probably energized by GTP. The isolation of the GTP-dependent acylCoA synthetase from sonicated kidney mitochondria supports this hypothesis.Oleate oxidation which is insensitive to phosphate and arsenate and requires oligomycin, utilizes "endogenous" ATP and is not linked to added carnitine.The third oleate oxidation system requires added ATP, CoA and phosphate and depends on a carnitine-linked transport mechanism.The prcsence of three acyl-CoA synthesizing systems have been postulated in intact rat liver mitochondria [l-71. The first one depends on substrate level phosphorylation and is inhibited by phosphate, arsenite and arsenate [1,2]. This system has been shown to be identical [I, 21 to the GTP-dependent acyl-CoA synthetase described both in beef liver IS] and in rat liver mitochondria [9]. The second system would derive the energy from "endogenous" ATP [1][2][3][4]6,7] and is thought to be "within" the mitochondrion. Acyl-CoA synthesized by this system does not depend on a carnitine-linked transport mechanism [lo] for its oxidation. The third acyl-CoA synthetase system would derive its energy from "exogenous') ATP C5-71 and is thought to occur "externally" or a t the surface of the mitochondrion. In this case, acyl-CoA must be converted to the carnitine ester for acyl transport to the "internal" site of p-oxidation.Van den Bergh [ill suggested that the acyl-Con synthesizing system which derives its energy from "endogenous" ATP is present in liver mitochondria, whereas in both kidney and heart mitochondria free long-chain fatty acids are oxidized only in the presence of added carnitine. Bode and Klingenberg [12] proposed that the activation of long-chain fatty acids in muscle mitochondria involves the transfer of carnitine from acetyl -carnitine to acyl-residues.Furthermore, no GTP-linked activation would be present in kidney 1113, heart 1111 and brain mitochondria [12].I n the present communication we report experiments which are relevant to the problem of longchain fatty acid oxidation in rat kidney mitochondria. Oxygen uptake was measured with a Clark oxygen electrode as described by Kielley and Bronk LlS].Acyl-CoA synthetase and succinic acid thiokinase were routinely assayed by measuring the rate of CoA sulfhydryl group disappearance [16]. The GTPdependent acyl-CoA synthetase and the succinic acid thiokinase were tested in a system containing 0.4 M Tris-HC1, pH 7.4, 0.3 M sucrose, 5 mM KBH,, 5 mM MgCl,, 2.5 mM GTP, 1 mM CoA and either 25 mM potassium octanoate, 2.5mM sodium oleate or 125mM sodium succinate. The total volume was 0.2 ml. The activity of the ATP-dependent acyl-CoA synthetase was measured in the presence of 5 mM ATP under similar conditions except that the Tris buffer was 50 mM and sucrose was repl...

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Direct and ¹⁸O-Exchange Measurements Relevant to Possible Activated or Phosphorylated States of Myosin^*

Cited by 55 publications

References 28 publications

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

High‐energy phosphate metabolism and energy liberation associated with rapid shortening in frog skeletal muscle

Organization of Fatty Acid Oxidation in Rat Kidney Mitochondria

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Direct and 18O-Exchange Measurements Relevant to Possible Activated or Phosphorylated States of Myosin*

Cited by 55 publications

References 28 publications

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

High‐energy phosphate metabolism and energy liberation associated with rapid shortening in frog skeletal muscle

Organization of Fatty Acid Oxidation in Rat Kidney Mitochondria

Contact Info

Product

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Direct and ¹⁸O-Exchange Measurements Relevant to Possible Activated or Phosphorylated States of Myosin^*