Equilibrium Enzymes in Regulatory Systems

Abstract

See related article, p 68–73 The work by Karamat et al1 contains evidence that creatine kinase (CK) mRNA in vascular smooth muscle has a direct correlation with blood pressure. This result is both interesting and problematic: there is no previous evidence from in vitro experiments that smooth muscle CK is a regulatory enzyme, and there is considerable evidence that the enzyme is at equilibrium inside cells. As the data indicate a regulatory role for the equilibrium enzyme CK in control of blood pressure, the general problem of investigating the role of an equilibrium enzyme in a regulatory system is inherently intriguing. Cytosolic CK has been always considered an equilibrium enzyme, whether in striated or smooth muscle. This assumption was used to calculate the free concentration of ADP in cells, in which the concentrations of ATP, PCr, and creatine could be determined. Phosphate NMR was used to measure the relative free concentrations of ATP, PCr, and Pi, and from the chemical shifts of Pi and β-ATP, the pH and free Mg++ of the cell could also be determined. Coupled with chemical measurements of creatine, and using in vitro measurements of the CK equilibrium constant, the free concentration of ADP (and thus the free energy of the cell) could be calculated. Measurements of free ADP cannot be made from chemical analyses because ADP liberated from f-actin compromises these measurements.2 ADP could not be seen as a peak in striated muscle phosphate NMR because of both its low concentration and chemical shift similarity to the ATP resonances. The lower concentration of Mg++ in smooth muscle meant that β-ADP had a different chemical shift from γ-ATP, and ADP peaks could be seen under …