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 autoproteolytic 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 immunodetection 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.
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November 1996
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Research Article|
November 15 1996
Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots Available to Purchase
Franck JAMES;
Franck JAMES
*Institut National de la Recherche Agronomique, Station de Physiologie Végétale, BP 81, F-33883 Villenave d'Ornon Cedex, France
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Renaud BROUQUISSE;
Renaud BROUQUISSE
‡
*Institut National de la Recherche Agronomique, Station de Physiologie Végétale, BP 81, F-33883 Villenave d'Ornon Cedex, France
‡To whom correspondence should be addressed.
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Claude SUIRE;
Claude SUIRE
†Institut de Biochimie et de Génétique Moléculaire, CNRS, UPR 9026, 1 rue Camille Saint-Saens, 33077 Bordeaux Cedex, France
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Alain PRADET;
Alain PRADET
*Institut National de la Recherche Agronomique, Station de Physiologie Végétale, BP 81, F-33883 Villenave d'Ornon Cedex, France
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Philippe RAYMOND
Philippe RAYMOND
*Institut National de la Recherche Agronomique, Station de Physiologie Végétale, BP 81, F-33883 Villenave d'Ornon Cedex, France
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Publisher: Portland Press Ltd
Received:
March 28 1996
Revision Received:
July 15 1996
Accepted:
August 01 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 320 (1): 283–292.
Article history
Received:
March 28 1996
Revision Received:
July 15 1996
Accepted:
August 01 1996
Citation
Franck JAMES, Renaud BROUQUISSE, Claude SUIRE, Alain PRADET, Philippe RAYMOND; Purification and biochemical characterization of a vacuolar serine endopeptidase induced by glucose starvation in maize roots. Biochem J 15 November 1996; 320 (1): 283–292. doi: https://doi.org/10.1042/bj3200283
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