Isolation of Intact Protein Storage Vacuoles from Barley Aleurone (Identification of Aspartic and Cysteine Proteases)
- 1 February 1996
- journal article
- Published by Oxford University Press (OUP) in Plant Physiology
- Vol. 110 (2) , 521-529
- https://doi.org/10.1104/pp.110.2.521
Abstract
Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). We developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. Isolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. We used isolated PSV to identify and characterize PSV-associated proteolytic activities. Isolated PSV contained cysteine proteases and aspartic proteases (APs). Gibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vulgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. Immunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. We conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.Keywords
This publication has 14 references indexed in Scilit:
- Purification and Characterization of AleurainPlant Physiology, 1992
- In Vitro Processing of Aleurain, a Barley Vacuolar Thiol Protease.Plant Cell, 1990
- A Major Gibberellic Acid-Induced Barley Aleurone Cysteine Proteinase Which Digests HordeinPlant Physiology, 1990
- A Proteinase from Germinating BarleyPlant Physiology, 1988
- Purification and Characterization of Gibberellic Acid-Induced Cysteine Endoproteases in Barley Aleurone LayersPlant Physiology, 1988
- Hormonal Regulation of the Development of Protease and Carboxypeptidase Activities in Barley Aleurone LayersPlant Physiology, 1986
- Isolation and Characterization of Oat Aleurone and Starchy Endosperm Protein BodiesPlant Physiology, 1983
- Enzymes Associated with Protein Bodies Isolated from Ungerminated Barley SeedsPlant Physiology, 1969
- Gibberellic Acid-Induced Synthesis of Protease by Isolated Aleurone Layers of BarleyPlant Physiology, 1967
- A test for de novo synthesis of enzymes: density labeling with H2O18 of barley alpha-amylase induced by gibberellic acid.Proceedings of the National Academy of Sciences, 1967