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• Articles by E.J. Potma
• Articles by G.J. Stienen

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• Effects of pH on myofibrillar ATPase activity in fast and sl...

  Effects of pH on myofibrillar ATPase activity in fast and slow skeletal muscle fibers of the rabbit Biophysical Journal, Volume 67, Issue 6, 1 December 1994, Pages 2404-2410 E.J. Potma, I.A. van Graas and G.J. Stienen Abstract In permeabilized single fibers of fast (psoas) and slow (soleus) muscle from the rabbit, the effect of pH on isometric myofibrillar ATPase activity and force was studied at 15 degrees C, in the pH range 6.4–7.9. ATPase activity was measured photometrically by enzymatic coupling of the regeneration of ATP to the oxidation of NADH, present in the bathing solution. NADH absorbance at 340 nm was determined inside a measuring chamber. To measure ATP turnover in single soleus fibers accurately, a new measuring chamber (volume 4 microliters) was developed that produced a sensitivity approximately 8 times higher than the system previously used. Under control conditions (pH 7.3), the isometric force was 136 and 115 kN/m2 and the ATP turnover was 0.43 and 0.056 mmol per liter fiber volume per second in psoas and soleus fibers, respectively. Over the pH range studied, isometric force increased monotonically by a factor 1.7 for psoas and 1.2 for soleus fibers. In psoas the isometric ATPase activity remained constant, whereas in soleus it slightly decreased with increasing pH. The pH dependency of relative tension cost (isometric ATPase activity divided by force) was practically identical for psoas and soleus fibers. In both cases it decreased by about a factor 0.57 as pH increased from 6.4 to 7.9. The implications of these findings are discussed in terms of cross-bridge kinetics. For both fiber types, estimates of the reaction rates and the distribution of cross-bridges and of their pH dependencies were obtained. A remarkable similarity was found between fast- and slow-twitch fibers in the effects of pH on the reaction rate constants. Abstract | PDF (814 kb)

• Measurement of Nucleotide Exchange Rate Constants in Single ...

  Measurement of Nucleotide Exchange Rate Constants in Single Rabbit Soleus Myofibrils during Shortening and Lengthening Using a Fluorescent ATP Analog Biophysical Journal, Volume 78, Issue 2, 1 February 2000, Pages 918-926 Ibuki Shirakawa, Shigeru Chaen, Clive R. Bagshaw and Haruo Sugi Abstract The kinetics of displacement of a fluorescent nucleotide, 2′(3′)--[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5′-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (Chaen et al., 1997, 73:2033–2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The for Cy3-EDA-ATP and the for Cy3-EDA-ADP binding to the myofibril A-band were 1.9M and 3.8M, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6M and 50M, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045±0.007s (=32) at 10°C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066s (isometric) to 0.14s at 20°C with increasing shortening velocity up to 0.1 myofibril length/s (, the shortening velocity under no load was ∼0.2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16nm for a single cycle of the ATPase. Abstract | Full Text | PDF (618 kb)

• At Physiological Temperatures the ATPase Rates of Shortening...

  At Physiological Temperatures the ATPase Rates of Shortening Soleus and Psoas Myofibrils Are Similar Biophysical Journal, Volume 85, Issue 5, 1 November 2003, Pages 3132-3141 R. Candau, B. Iorga, F. Travers, T. Barman and C. Lionne Abstract We obtained the temperature dependences of the adenosine triphosphatase (ATPase) activities (calcium-activated and relaxed) of myofibrils from a slow muscle, which we compared with those from a fast muscle. We chose rabbit soleus and psoas because their myosin heavy chains are almost pure: isoforms I and IIX, respectively. The Arrhenius plots of the ATPases are linear (4–35°C) with energies of activation for soleus myofibrils 155kJmol (activated) and 78kJmol (relaxed). With psoas myofibrils, the energies of activation were 71kJmol (activated) and 60kJmol (relaxed). When extrapolated to 42°C the ATPase rates of the two types of myofibril were identical: 50s (activated) and 0.23s (relaxed). Whereas with psoas myofibrils the for adenosine triphosphate (activated ATPase) is relatively insensitive to temperature, that for soleus myofibrils increased from 0.3M at 4°C to 66.5M at 35°C. Our results illustrate the importance of temperature when comparing the mechanochemical coupling in different types of muscle. We discuss the problem of how to reconcile the similarity of the myofibrillar ATPase rates at physiological temperatures with their different mechanical properties. Abstract | Full Text | PDF (198 kb)

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Abstract

The influence of P(i) and pH was studied on myofibrillar ATP turnover and force development during maximally activated isometric contractions, in skinned single fibers from rabbit soleus and psoas muscle. ATP hydrolysis was coupled to the breakdown of NADH, which was monitored photometrically at 340 nm. In psoas the depression by phosphate of force is twice that of ATP turnover, but in soleus force and ATP turnover are depressed equally by P(i). Most, but not all, of the ATPase and force values observed for a combination of high P(i) and low pH could be explained by independent effects of P(i) and pH. The effects of P(i) and pH on ATP turnover can be understood by a three-state cross-bridge scheme. Mass action of phosphate on the reaction from the actomyosin(AM).ADP state to the AM.ADP.P(i) state may largely account for the phosphate dependencies of ATPase activity found. Protons affect cross-bridge detachment from the AM.ADP state and the rate of the AM.ADP.P(i)-to-AM.ADP transition. In this scheme, the effects of P(i) and pH on cross-bridge kinetics appeared to be largely independent.