IMP degradative capacity in rat skeletal muscle fiber types.

During contractions, when the rate of ATP hydrolysis exceeds that of ADP phosphorylation, inosine 5'-monophosphate (IMP) accumulates in skeletal muscle. If the cellular energy balance is not promptly restored, subsequent purine degradation to inosine via 5'-nucleotidase can occur, a process that is most robust in the slow-twitch red, as compared to fast-twitch, skeletal muscle. We measured the distribution of 5'-nucleotidase activity among membrane-bound and soluble fractions of fiber specific skeletal muscle sections and found most (80-90%) of the total 5'-nucleotidase activity to be membrane-bound.

Novel aspects of tetramer assembly and N-terminal domain structure and function are revealed by recombinant expression of human AMP deaminase isoforms.

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4 degreesC. Expression of N-truncated cDNAs (DeltaL96AMPD1 and DeltaM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E.

Cytoarchitectural and metabolic adaptations in muscles with mitochondrial and cytosolic creatine kinase deficiencies.

We have blocked creatine kinase (CK) mediated phosphocreatine (PCr) <==> ATP transphosphorylation in mitochondria and cytosol of skeletal muscle by knocking out the genes for the mitochondrial (ScCKmit) and the cytosolic (M-CK) CK isoforms in mice. Animals which carry single or double mutations, if kept and tested under standard laboratory conditions, have surprisingly mild changes in muscle physiology. Strenuous ex vivo conditions were necessary to reveal that MM-CK absence in single and double mutants leads to a partial loss of tetanic force output.

Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice.

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK-/-) or also deficient in mitochondrial creatine kinase (CK-/-) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK-/- and CK-/- mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 (AMPD1) gene expression was unchanged.

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion.

We examined a possible mechanism to account for the maintenance of peak AMP deamination rate in fast-twitch muscle of rats fed the creatine analog beta-guanidinopropionic acid (beta-GPA), in spite of reduced abundance of the enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined at saturating AMP concentration) and AMPD protein abundance (Western blot) were coordinately reduced approximately 80% in fast-twitch white gastrocnemius muscle by beta-GPA feeding over 7 wk. Kinetic analysis of AMPD in the soluble cell fraction demonstrated a single Michaelis-Menten constant (Km; approximately 1.