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. 2003 Jun 1;372(Pt 2):443–451. doi: 10.1042/BJ20021687

Biochemical and mass spectrometric characterization of soluble ecto-5'-nucleotidase from bull seminal plasma.

Carlo Fini 1, Fabio Talamo 1, Silvia Cherri 1, Marcello Coli 1, Ardesio Floridi 1, Lino Ferrara 1, Andrea Scaloni 1
PMCID: PMC1223402  PMID: 12608891

Abstract

Ecto-5'-nucleotidase (ecto-5'-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5'-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln(547) and/or Phe(548). Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn(403). Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5'-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

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