An aspartic proteinase present in seeds cleaves Arabidopsis 2 S albumin precursors in vitro.
The Arabidopsis thaliana 2 S albumins are examples of vacuolar proteins which undergo intensive posttranslational processing. An in vitro processing assay to screen for processing enzymes present in seeds was developed using an in vitro synthesized 2 S albumin precursor as the substrate. A protease was characterized which cleaved the substrate into two fragments with molecular weights (as determined from their migration distance on SDS-polyacrylamide gel) corresponding to those of the small and large subunits of Arabidopsis 2 S albumin. The pH optimum of this protease activity, its inhibition by pepstatin A, and partial sequence data led to the conclusion that the protease under study was an aspartic proteinase. Synthetic peptides representing two 2 S albumin propeptides allowed the determination of the in vitro cleavage sites, and suggested that the protease activity is capable, in vitro, of cleaving the amino-terminal propeptide as well as the internal propeptide linking the two subunits. Alterations of the amino acids in and around the cleavage sites, made to study the specificity of the protease activity, suggest that both structural and sequence determinants are important in cleavage site recognition.
D'Hondt
K
K
Laboratorium voor Fysiologische Scheikunde, Universiteit Gent, Belgium.
Bosch
D
D
Van Damme
J
J
Goethals
M
M
Vandekerckhove
J
J
Krebbers
E
E
D000418:Albumins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D000418:Albumins; D010455:Peptides; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases