PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF A VACUOLAR SERINE ENDOPEPTIDASE INDUCED BY GLUCOSE STARVATION IN MAIZE ROOTS

Citation
F. James et al., PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF A VACUOLAR SERINE ENDOPEPTIDASE INDUCED BY GLUCOSE STARVATION IN MAIZE ROOTS, Biochemical journal, 320, 1996, pp. 283-292
Citations number
54
Categorie Soggetti
Biology
Journal title
ISSN journal
02646021
Volume
320
Year of publication
1996
Part
1
Pages
283 - 292
Database
ISI
SICI code
0264-6021(1996)320:<283:PABOAV>2.0.ZU;2-B
Abstract
An endopeptidase (designated RSIP, for root-starvation-induced proteas e) was purified to homogeneity from glucose-starved maize roots. The m olecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing cond itions and 62 kDa by gel filtration on a Sephacryl S-200 column. The i soelectric point of RSIP was 4.55. The purified enzyme was stable, wit h no autoproteolytic activity. The enzyme activity was strongly inhibi ted by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a seri ne protease. The maximum proteolytic activity against different protei n substrates occurred at pH 6.5. With the exception of succinyl-Leu-Le u-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determinati on of the cleavage sites in the oxidized B-chain of insulin showed spe cificity for hydrophobic residues at the P2 and P3 positions, indicati ng that RSIP is distinct from other previously characterized maize end opeptidases. Both subcellular fractionation and immunodetection in sit u indicated that RSIP is localized in the vacuole of the root cells. R SIP is the first vacuolar serine endopeptidase to be identified. Gluco se starvation induced RSIP: after 4 days of starvation, RSIP was estim ated 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.