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The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

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Abstract

A proline iminopeptidase (EC. 3.4.11.5) was isolated from shoots of 3 day old seedlings. The purification procedure consisted of 5 steps: acid precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on Sepharose CL 6B, twice repeated hydrophoic chromatography on Phenyl-Sepharose HP. The enzyme was purified 404.8-fold, with the specific activity of 8.5 units mg−1 of protein with recovery yield of 3%. The purified enzyme had a molecular mass of 225 kDa estimated by gel filtration and 55.4 kDa by SDS PAGE. This indicates that native enzyme is composed of four subunits. The enzyme was specific for proline β-naphtylamide among various amino acid β-naphtylamides.

An optimal activity was observed at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The enzyme was strongly inhibited by pHMB, E-64, heavy metal ions and partially by PMSF, DFP. The results suggest that cysteine and serine residues may participate in the enzyme activity.

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Abbreviations

2-ME:

2-mercaptoethanol

PAGE:

polyacrylamide gel electrophoresis

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

EDTA:

ethylenediaminetetraacetic acid

E-64:

transepoxysuccinyl-L-leucyloamido-(4-guanidino)butane

pHMB:

p-hydroxymercuribenzoic acid

βNA:

β-naphthylamide

PMSF:

phenyl methylsulfonyl fluoride

DFP:

diisopropyl fluorophosphate

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Authors and Affiliations

  1. Department of Biochemistry, Warsaw Agricultural University, Nowoursynowska 159, 02-776, Warszawa, Poland

    Urszula Szawłowska, Wiktor Prus & Wiesław Bielawski

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  1. Urszula Szawłowska
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  2. Wiktor Prus
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  3. Wiesław Bielawski
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Correspondence to Urszula Szawłowska.

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Szawłowska, U., Prus, W. & Bielawski, W. The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.). Acta Physiol Plant 28, 517–524 (2006). https://doi.org/10.1007/s11738-006-0047-5

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  • DOI: https://doi.org/10.1007/s11738-006-0047-5

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