Abstract
Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).
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Abbreviations
- RP:
-
reversed phase
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- TFA:
-
trifluoroacetic acid
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Dedicated to the memory of Professor Juhani Mikola who started this study before his untimely death in September 1988. A preliminary report of the results was presented at the 15th Congress of the Scandinavian Society for Plant Physiology, Turku, Finland (Siuro et al. 1988)
This study was supported by grants from Foundation for Biotechnical and Industrial Fermentation Research. We are grateful to Professor Leevi Kããriãinen and Professor Mart Saarma for their encouraging support after the death of Professor Juhani Mikola. Studies with aspartic-proteinase inhibitors were carried out during a visit (P.S.) to the Department of Biochemistry, University College, Cardiff, Wales, UK. We greatly acknowledge Professor John Kay for making this part of the study possibly and Dr. Anthony D. Richards for generous help with the experiments. We thank Tiina Aho-Mantila and Pirjo Kauppinen for skilled technical assistance, and Professor Tuomas Sopanen and Dr. Alan Schulman for critical reading of the manuscript.
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Sarkkinen, P., Kalkkinen, N., Tilgmann, C. et al. Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D. Planta 186, 317–323 (1992). https://doi.org/10.1007/BF00195311
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DOI: https://doi.org/10.1007/BF00195311