Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart

Abstract

Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.

The influence of phosphatidate bilayers on pig heart AMP deaminase. Crucial role of pH-dependent lipid-phase transition

“…Orthophosphate is an effector known to exhibit a negative allosteric effect on AMP deaminase from skeletal muscle, brain [18] and pig heart [8]. In our experiments 10 mMorthophosphate exerted a 50 % inhibitory effect on pig heart AMP deaminase (Fig.…”

“…2.). Orthophosphate, known as a negative allosteric effector of AMP deaminase [8], inhibits the reaction by three orders of magnitude less, the half inhibition being reached at about 10-3 M-phosphate (Fig. 3).…”

“…Liposomes containing different kinds of phospholipids were prepared as described previously [8], with slight modifications. A stream of argon instead of nitrogen was used throughout the preparation of liposomes containing dioleoylphosphatidate.…”

“…The resulting lipid dispersions were freshly prepared every day. Enzyme preparation AMP deaminase from pig heart was purified by cellulose-phosphate-affinity chromatography as previously described [8]. Enzyme assay…”

“…AMP deaminase from pig heart is a cytosolic enzyme which may be the subject of a subtle interplay of various regulatory factors, including purine nucleotides, orthophosphate and lipid membranes. Interaction of this enzyme with phosphatidylcholine-containing'membranes was shown to enhance the activating effect of ATP and ADP [8]. Cardiolipin was found to inhibit the activity of AMP-deaminase, whereas a complex mixture of natural phospholipids isolated from heart cells, displayed an activating effect [9].…”