The catalytic activity of adenosine monophosphate deaminase in murine thymus as revealed by Transmission Electron Microscopy

Abstract

AMP deaminase was visualized in a manner analogous to our previous approach with adenosine deaminase. The caloro-analog of AMP, i.e., 6 Chloropurine riboside 5′ monophosphate (CPRMP) (from Sigma) was shown to be a substrate of this enzyme which liberates Cl− which is precipitated with added Ag+, and, after exposure to light electron sense Ag° grains are deposited at loci of enzyme activity. The substrate at a concentration of 1.1 mM in 50 mM HEPES buffer, pH 7.2, in the presence of 150 mM K+ (as the acetate), 3mM ATP and 10μM pentostatin deoxycoformycin) was incubated with freshly excised tissue from a female C-57 BL/6 mouse. The substrate concentration is 1.4 times Km for rabbit muscle enzyme (Sigma), and K+ and ATP are allosteric activators of this Enzyme. Because many cells contain ecto 5′-nucleotidase activity, pentostatin was added to prevent manifestation of denosine deaminase activity of dechlorination of any nucleoside that might be formed. Any Cl− formed by adenosine deaminase would be precipitated with the added Ag+ and thus result in Ago deposits which would be indistinguishable from the Ago deposited by AMP deaminase.