Comparative Enzymology of Muscle Proteins: Role of Salvage Synthesis of Adenylate Nucleotides in Eukaryotic Cells

I have a particular interest in the comparative enzymology of muscle proteins. The primary focus has been on the enzyme AMP deaminase (AMPD). AMPD is expressed as several tissue- specific and developmentally-regulated isoforms, reflecting a multigene fa mily. In mature vertebrates, the major fraction of this activity is expressed in skeletal muscle as a single isoform (AMPD.M). It is postulated that during exercise, the coordinate activities of AMPD.M and myokinase help to maintain a high ratio of [ATP ]/[ADP]. Depletion in the adenine nucleotide pool during intense contractions is accompanied by production of ammonia and IMP via the effectively irreversible action of AMPD.M; IMP is reconverted to AMP via the sequential action of adenylos uccinate synthetase and adenylosuccinase, thus completing the purine nucleotide cyle.
Fig. 1 --- Comparison of CNBr Fragments of AMPD.M from Chicken and Rabbit with the Predicated Homologous Segments of the Enzymes from Human and Rat

The sequence of the first twenty-five residues of the 17-kDa CNBr fragments of the rabbit and chicken enzymes are illustrated and compared at the top of the figure. The partial sequences of the rat and human enzymes are based on the polypeptides predicte d from the gene sequences reported by Sabina et al.[J. Biol.Chem. 262, 12397-12400 (1987) and Neurology 42, 170-179 (1992), respectively. Estimates of homology are based on BESTFIT analysis using the local homology algorithm of Smith and Waterman.

A recent manifestation of our interest in this enzyme is reflected in analysis of the primary sequences of cyanogen bromide fragments of AMPD.M from rabbit and chicken muscle (Fig. 1). The apparent size (87.5-kDa) of the major polypeptide in freshly isol ated chicken breast muscle AMPD.M was comparable to that predicted from the sequences of the genes for the major muscle isoforms (AMPD1) from human and rat. The size of the subunit of AMPD from chicken muscle is indistinguishable from that of the rabbit enzyme. The peptide profiles of cyanogen bromide digests of AMPD.M from chicken and rabbit share a 17-kDa fragment, representing approximately 20% of the intact subunits of these enzymes. The first twenty-five residues of these fragments are 88.5% ident ical; the rabbit and chicken segments are greater than 92% and 84% identical, respectively, to the sequences predicted for residues 310 to 335 for AMPD.M from human and rat. Polyclonal rabbit antisera directed against AMPD.M from chicken breast recognize the full length AMPD.M polypeptides on immunoblots of extracts of both avian and rabbit muscle, including an antiserum from rabbit in which the antibody wa prepared. The 17-kDa fragments, derived by incomplete cleavage of highly conserved internal segme nts of the deaminase subunit, share epitopes involved in the autorecognition of rabbit AMPD.M by rabbit polyclonal antibodies directed against the avian AMPD.

I also have a long standing interest in the regulation of adenylate nucleotide metabolism in eukaryotic cells, particularly the role of base and nucleoside reutilization in maintenance of energy charge in cells which lack the capacity for de novo p urine biosynthesis. These considerations led to the demonstration of a high capacity for adenine salvage by avian erythrocytes and the role of the salvage enzyme, adenine phosphoribosyltransferase (APRT) in these cells.

Evidence was obtained by colleagues (Dan Kohl and Georgia Shearer) that under stress conditions, proline may be an important source of energy for nitrogen fixation. This observation, together with an apparent intimate relationship between de novo biosynthesis of purine nucleotides, proline metabolism, and assimilation of fixed nitrogen in soybean nodules, led us to explore certain aspects of the enzymology of proline biosynthesis in nodule tissue.

More recently, this developing involvement in plant biochemistry, together with a continuing interest in the regulation of purine metabolism has let to consideration of the role of salvage synthesis of adenylate nucleotides in tissues and root nodules of mature and developing soybean plants.

While less well characterized than comparable pathways in animal systems, the potential impact of the purine salvage pathways in plants is dramatically illustrated by the observation that Arabidopsis thaliana plants deficient in APRT, are male ster ile.

It seems likely that salvage of adenine and adenosine potentially contributes to, and is perhaps crucial for, the maintenance of energy charge, production of essential cofactors and nucleic acid precursors, and modulation of the levels of active cytokinin s. These salvage pathways may also contribute to assimilation of fixed nitrogen.