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Biochim Biophys Acta. 2000 Jun 15;1479(1-2):37-51.

Distinct physical and structural properties of the ovine uterine serpin.

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  • 1Department of Animal Sciences, University of Florida, Gainesville 32610-0920, USA.

Abstract

Experiments were performed to examine the relationship between the structure and function of ovine uterine serpin (OvUS). Limited proteolytic digestion of OvUS caused cleavage of the 55-57 kDa OvUS to a 42 kDa product nearly identical in molecular weight to a naturally-occurring breakdown product of OvUS. N-terminal amino acid sequencing and MALDI-MS revealed that, unlike other serpins, OvUS was preferentially cleaved at about 70 amino acids upstream of the putative reactive center loop. Analysis of the partially-digested protein by gel filtration chromatography suggested that the C-terminal fragment of the protein was still associated under nondenaturing conditions. Partial digestion of OvUS had no effect on the protein's secondary structure, thermal stability, ability to bind lymphocytes or pepsin, or inhibitory activity towards pepsin or mitogen-induced lymphocyte proliferation. In contrast, mild denaturation of OvUS with 0.5 M guanidine HCl increased thermal stability. Unlike for other serpins, the increase in thermal stability was lost upon removal of the denaturant. Incubation of OvUS with 100 fold molar excess of a peptide corresponding to the putative P(14)-P(2) region of the RCL for 24 h at 37 degrees C to induce binary complex formation had no effect on its secondary structure and did not alter the biological activity of the protein. Synthetic peptides corresponding to the putative P(14)-P(2) region and the P(7)-P(15') region of the RCL were not inhibitory to pepsin activity or lymphocyte proliferation. Taken together, these results indicate that the conformation of OvUS is distinct from the prototypical serpin because conditions that lead to the large-scale conformational change in other serpins such as antithrombin III and alpha(1)-antitrypsin do not cause similar changes in OvUS. Moreover, the putative RCL does not seem to contain the activity required to inhibit lymphocyte proliferation or pepsin activity.

PMID:
11004528
[PubMed - indexed for MEDLINE]
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