Abstract
Palpable breast cysts with an apocrine epithelial lining (type 1) are reported to be associated with a higher risk of developing breast cancer. The composition of breast cyst fluid (BCF) might include those factors involved in this increased risk. In this study peptidase activities that were active against the substrate [125I]metenkephalin-Arg-Phe were detected in BCF. The products were identified by reversed phase high-performance liquid chromatography (HPLC) as [125I]Tyr-Gly-Gly and [125I]Met-enkephalin. This proteolysis was not inhibited by PCMB, pepstatin A, leupeptin or aprotinin but was by EDTA, showing that the activity was due to metalloproteases. The production of [125I]Try-Gly-Gly was inhibited by phosphoramidon and thiorphan, whereas that of [125I]met-enkephalin was inhibited by captopril and Bothrops jararaca peptide, indicating that these activities are enkephalinase and angiotensin-converting enzyme (ACE) respectively. A fluorometric assay for ACE demonstrated that ACE levels are significantly higher in type 2 BCF than in type 1 BCF (30.8 vs 6.1 nmol hr-1 10 microliters-1, P < 0.001). As the increased risk of cancer is linked to type 1 cysts it is possible that higher levels of peptidase in type 2 BCF reflect a protective environment in the breast in which mitogenic peptide growth factors are neutralised by proteolysis.
Highlights
As the increased risk of cancer is linked to type 1 cysts it is possible that higher levels of peptidase in type 2 breast cyst fluid (BCF) reflect a protective environment in the breast in which mitogenic peptide growth factors are neutralised by proteolysis
In this paper we describe the characterisation of the peptidase activities in BCF that cleave Met-enkephalin-Arg-Phe
[125I]Met-enkephalin-Arg-Phe eluted as a single radioactive peak on reverse-phase high-performance liquid chromatography (HPLC) (Figure la), after incubation of this peptide with a type 1 breast cyst fluid two further peaks were observed that eluted in the positions of the [1251I]met-enkephalin and the [1251I]Tyr-Gly-Gly markers (Figure lb)
Summary
Breast cyst fluid was obtained by needle aspiration of breast cysts from women attending the breast clinic at St Mary's Hospital, London, with their informed consent. Each set of incubations included a control that contained no breast cyst fluid. Incubated with 100,l of 2% o-phthaldialdehyde for 15 min at room temperature and this reaction terminated with 200 pl of 3.0 M hydrochloric acid. The interassay coefficient of variation was 3.8%, determined by including aliquots of the same BCF in assays over a period of 3 months. This demonstrated that the activity was stable on storage despite freezing and thawing. Substrates, rabbit lung ACE and inhibitors were. Bothrops jararaca peptide is available as ACE inhibitor peptide from Sigma and is a proline-rich peptide (Ondetti et al, 1971). Mann- Whitney test and correlation coefficients were calculated using Spearman's rank correlation method
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