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: F James, R Brouquisse, C Suire, A Pradet, P Raymond
• Journal: The Biochemical journal (Biochem J) Vol. 320 ( Pt 1) Pg. 283-92 (Nov 15 1996) ISSN: 0264-6021 [Print] England
• PMID: 8947499 (Publication Type: Journal Article)

Chemical References

• Insulin
• Serine Proteinase Inhibitors
• Endopeptidases
• Serine Endopeptidases
• serine endopeptidase
• Glucose

Topics

• Amino Acid Sequence
• Endopeptidases (isolation & purification, metabolism)
• Enzyme Stability
• Glucose
• Hydrogen-Ion Concentration
• Hydrolysis
• Insulin (chemistry, metabolism)
• Kinetics
• Molecular Sequence Data
• Plant Roots (enzymology)
• Serine Endopeptidases
• Serine Proteinase Inhibitors (pharmacology)
• Subcellular Fractions (enzymology)
• Substrate Specificity
• Vacuoles (enzymology)
• Zea mays (enzymology)