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

Abstract

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 °C. Expression of N-truncated cDNAs (ΔL96AMPD1 and ΔM90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher K(m(app)) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4 °C. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggl, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform- specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.