title: Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots.
abstract: An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation.
authors.affiliation: Institut National de la Recherche Agronomique, Station de Physiologie, Végétale Villenave d'Ornon, France.
authors.forename: F
authors.initials: F
authors.lastname: James
authors.forename: R
authors.initials: R
authors.lastname: Brouquisse
authors.forename: C
authors.initials: C
authors.lastname: Suire
authors.forename: A
authors.initials: A
authors.lastname: Pradet
authors.forename: P
authors.initials: P
authors.lastname: Raymond
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005947:Glucose; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007328:Insulin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000201:enzymology*; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology*
chemical_list: D007328:Insulin; D015842:Serine Proteinase Inhibitors; D010450:Endopeptidases; D012697:Serine Endopeptidases; D005947:Glucose