Purification, biochemical characterisation, and mass spectrometry analysis of phenylalanine aminopeptidase from the shoots of pea plants

• Autorzy: Pyrzyna M. , Szawlowska U. , Bielawski W. , Zdunek-Zastocka E.
• Języki publikacji: EN

Abstrakty

EN

Phenylalanine aminopeptidase (Phe-AP) was isolated from the shoots of 3-week-old pea plants and purified to molecular homogeneity using a four-step purification procedure (ammonium sulphate precipitation, chromatography on DEAE-cellulose, phenyl-sepharose HP, and Protein-Pak Q 8HR HPLC columns). The enzyme was purified 513-fold with a recovery of 8%. The molecular weight of the purified enzyme as determined by SDSPAGE and gel filtration was approximately 60 kDa, and the enzyme appeared to be a monomer. Its pH and temperature optimum were pH 7.5 and 37°C, respectively. The enzyme prefers substrates with Phe at the N-terminus, although a high activity for substrates with N-terminal Tyr, Trp, Leu, and Met was also observed. The activity with Leu-β-naphthylamide was at least two times lower than that with Phe-β-naphthylamide (Phe-β-NA). The Km value for activity with Phe-β-NA was the lowest amongst the substrates tested, and it was 7.5 × 10⁻⁵ M. The activity of Phe-AP was not inhibited by EDTA, 1,10-phenanthroline and pepstatin A. The most effective inhibitors were pHMB and E-64, which modify sulphydryl groups; however, a significant inhibition in the presence of DFP and PMSF, both of which are serine protease inhibitors, was also observed. By applying mass spectrometry analysis, the peptides derived from the purified Phe-AP were assigned to amino acid sequences of the leucine aminopeptidases of N-type (LAPs-N).

• Wydawca: -
• Czasopismo: Acta Physiologiae Plantarum
• Rocznik: 2011
• Tom: 33
• Numer: 2
• Opis fizyczny: p.609-617,fig.,ref.

Twórcy

autor

Pyrzyna M.

• Department of Biochemistry, Warsaw University of Life Sciences, SGGW, Nowoursynowska 159, Warsaw 02-776, Poland

autor

Szawlowska U.

• Department of Biochemistry, Warsaw University of Life Sciences, SGGW, Nowoursynowska 159, Warsaw 02-776, Poland

autor

Bielawski W.

• Department of Biochemistry, Warsaw University of Life Sciences, SGGW, Nowoursynowska 159, Warsaw 02-776, Poland

autor

Zdunek-Zastocka E.

• Department of Biochemistry, Warsaw University of Life Sciences, SGGW, Nowoursynowska 159, Warsaw 02-776, Poland

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