A comparative study of the expression of serine proteinases in quiescent seeds and in developing Canavalia ensiformis plants.
An alkaline proteinase activity is present in quiescent seeds and up to the 24th day of development of Canavalia ensiformis DC (L.) plants. By a simple protocol consisting of cation exchange chromatography, followed by an anion exchange column, a serine proteinase (Q-SP) was purified to homogeneity from quiescent seeds. Q-SP consists of a 33 kDa chain with an optimum pH between 8.0 and 9.0. Arginine residues at P1 and P2 subsites favour binding to the substrate, as shown by the KM assay with N-alpha-benzoyl-DL-arginine-4-nitroanilide-hydrochloride and N-benzoylcarboxyl-L-arginyl-L-arginine-7-amido-4-methylcoumarin. The same protocol was used for partial purification of benzamidine-sensitive enzymes from the developing plant. On the 7th day, a new benzamidine-sensitive enzyme is synthesized in the seedling, seen as the second active peak appearing in anion exchange chromatography. A benzamidine-sensitive enzyme purified from cotyledons presented a similar gel filtration profile as Q-SP, although it was eluted at different salt concentrations in the anion exchange chromatography. None of the enzymes was inhibited by PMSF, APMSF, or SBTI, but they were inactivated by benzamidine, TLCK, and leupeptin. Q-SP did not cleave in vitro C. ensiformis urease, concanavalin A, or its main storage protein, canavalin. In conclusion, a ubiquitous benzamidine-sensitive proteolytic activity was found in C. ensiformis from quiescent seeds up to 24 d of growth, which apparently is not involved in the hydrolysis of storage proteins and might participate in an as yet unidentified limited proteolysis event.
Demartini
Diogo Ribeiro
DR
Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Federal University of Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43422, Porto Alegre, RS 91501-970, Brazil.
Wlodawer
Alexander
A
Carlini
Célia Regina
CR
D001550:Benzamidines / Q000494:pharmacology; D032449:Canavalia / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D002845:Chromatography; D018548:Cotyledon / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D018517:Plant Roots / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D012639:Seeds / Q000187:drug effects / Q000201:enzymology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology
D001550:Benzamidines; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; C032157:benzamidine