Substrate preference of 5-nucleotidase and the pathways of AMP degradation in rat red skeletal muscle

Abstract

Adenosine can be formed from cytosolic AMP by 5)-nucleotidase (AMP-ND) or extracellular AMP via the ecto-enzyme. A 5)-nucleotidase (ND) also exists in an IMP-specific form (IMP-ND), and its substrate preference has not been characterized in oxidative skeletal muscle. Also, AMP deaminase (AMPD) competes with AMP-ND by using AMP as a substrate to form IMP by deamination. Again, the pathway dominating AMP metabolism in red skeletal muscle has not been elucidated. Therefore, the purpose of the study was to investigate the substrate specificity of ND and the metabolic pathways of AMP degradation in rat soleus muscle. The membrane and cytosolic fractions of muscle tissues were separated by differential centrifugation. The influence of AMPD on the pathways of AMP degradation by ND was investigated by inhibiting AMPD with coformycin (CFM), and the properties of AMP- and IMP-ND were studied by comparing the kinetic parameters of the enzymes. The data show that: 1) the preference for AMP over IMP for membrane-bound ND is about 1.8-fold of that for cytosol-bound which exhibits similar selectivity for AMP and IMP, and 2) the presence of CFM increases the maximal activity (Vmax) of cytosolic AMP-ND by 24% but has no effect on the Vmax of membrane-bound AMPND. These results suggest that in rat oxidative skeletal muscle, the membrane-bound ND has preference for AMP over IMP while the cytosol-bound does not, and AMPD affects the formation of adenosine only in cytosolic fractions.