Muscle 5′-Adenylic Acid Aminohydrolase: KINETIC PROPERTIES OF RAT MUSCLE ENZYME TREATED WITH PYRIDOXAL 5′-PHOSPHATE

Abstract The treatment of purified rat muscle AMP deaminase with pryidoxal-5'-P produces a decrease of the enzyme activity which is time- and pyridoxal-5'-P concentration-dependent. The reaction is reversible and can be changed to an irreversible reaction by reduction with NaBH4. The reduced pyridoxal-5'-P-AMP deaminase complex shows an absorption spectrum which is typical for pyridoxamine derivatives. Chromatographic analyses confirm the formation of a Schiff base between pyridoxal-5'-P and the e-amino group of lysine residues in the enzyme. The loss of activity is not a simple inactivation process due to modification of the catalytic site of the enzyme. GTP and ATP, which are allosteric inhibitors of AMP deaminase, show a protective effect against pyridoxal-5'-P inactivation whereas KCl at high concentration exerts a little protection and Pi and P-creatine are ineffective. The reduced pyridoxal-5'-P-AMP deaminase derivative with 30 to 35% residual activity and 6 to 7 moles of pyridoxal-5'-P bound per mole of protein has different kinetic and regulatory properties from the native enzyme: Vmax is the same but the Km value is increased; the enzyme shows a homotropic cooperativity also in the presence of activators, K+ and ADP, and is no longer sensitive to GTP, ATP, and high salt concentration inhibitions. Pi and P-creatine still exert an inhibitory effect. These kinetic properties are similar to those of the native enzyme in the presence of nucleoside triphosphates. The reduced pyridoxal-5'-P-AMP deaminase derivative prepared in the presence of GTP is still sensitive to inhibition by nucleoside triphosphates. It is likely that pyridoxal-5'-P binds at or near the binding sites for these compounds

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays