Effect of ionic strength and pH on the activity of pyruvate dehydrogenase complex from pig kidney cortex.
The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex is sensitive to changes in ionic strength (mu). At low ionic strength (mu = 0.04 M) the specific activity of PDC was 12.22 mumol/min/mg, whereas at high ionic strength (mu = 0.15 M) the measured activity of the complex decreased to 4.88 mumol/min/mg. The optimum activity of PDC was achieved within a small range of ionic strength, mu = 0.035-0.040 M. Increasing the ionic strength from mu = 0.05 to mu = 0.15 M decreased the s0.5 for pyruvate from 125 to 72 microM and increased the Hill coefficient from 1.0 to 1.3. The effect of pH on PDC activity also was dependent upon ionic strength. At pH 7.2 the activity of PDC at mu = 0.05 and mu = 0.15 M was 90 and 55% of the maximal activity, respectively. Furthermore, the effects of Na+, K+, HCO3-, Cl-, and HPO4(2-) on PDC activity were dependent on ionic strength and pH. The addition of K+ (80 mM) at mu = 0.10 and mu = 0.15 M increased the activity of PDC by 12 and 42%, respectively. Lowering the pH from 8.2 to 7.5 resulted in a decrease in the s0.5 for pyruvate from 179 to 110 microM and from 110 to 35 microM in the presence and absence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), and HPO4(2-) (10 mM), respectively. The observed changes in the properties of PDC in response to changes in ionic strength likely was a result of changes in the intramolecular electrostatic interactions within the complex. In this regard it was determined using two-dimensional agarose gel electrophoresis of the intact multienzyme complex that increasing the ionic strength to which PDC is exposed decreased the measured radius of PDC and may have decreased the electronegative surface charge of the complex.
Pawelczyk
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Easom
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RA
Olson
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MS
D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D009994:Osmolar Concentration; D010710:Phosphates / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D013552:Swine
D001639:Bicarbonates; D002712:Chlorides; D010710:Phosphates; D011768:Pyruvate Dehydrogenase Complex; D012964:Sodium; D011188:Potassium