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Plant Physiology 94:251-258 (1990)
© 1990 American Society of Plant Biologists

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Metabolism and Enzymology

A Major Gibberellic Acid-Induced Barley Aleurone Cysteine Proteinase Which Digests Hordein 1

Purification and Characterization

Susan M. Koehler2 and Tuan-Hua David Ho

Department of Biology, Plant Biology Program, Washington University, St. Louis, Missouri 63130, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Missouri 63130

We previously described the purification and characterization of a 37,000 Mr cysteine proteinase, designated EP-A, from gibberellic acid (GA3)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent Mr of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 Mr as determined by gradient SDS-PAGE.

2 Current address: USDA/ARS, Soybean and Alfalfa Laboratory, Room 328, Building 001, BARC-West, Beltsville, MD 20705.

1 Support by National Science Foundation grant DCB-8702299.




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