Bioassay for the determination of the intra- and extracellular activity of aminopeptidases in immobilized tomato cells.
Permeabilized tomato cells were cross-linked with glutaraldehyde in the absence of a carrier. The immobilized cells demonstrated significantly lower aminopeptidase (AP) activities than untreated control cells. However, when immobilized with pectate and alginate gels, the tomato cells retained their AP activities. A new method for the determination of the activity of both extra- and intracellular AP was developed, based on enzyme-catalyzed hydrolysis of a series of synthetic beta-naphthylamides (betaNA) of the L-amino acids Ala, Arg, Leu, Pro, Tyr, or of the synthetic beta-methoxynaphthylamides (betaMNA) of Ala and Arg. Extracellular AP--produced by calli, cell-suspension culture, or seedlings of tomato cells grown on agar--hydrolyzed these peptidic substrates to the free naphthalene amines and amino acids. Staining with Fast Garnet GBC salt under formation of bright reddish azo dyes readily allowed the determination of AP activities. For the tomato-cell suspension, the intracellular activity accounted for 91.3-93.9% of the total activity, and the extracellular one for 6.1-8.7%, respectively. Our method permits the rapid, simple, and specific determination of plant aminopeptidases.
Stano
Ján
J
Garden of Medicinal Plants, Faculty of Pharmacy, Comenius University, Odbojárov 10, SK-832 32 Bratislava, Slovakia.
Micieta
Karol
K
Tintemann
Herbert
H
Neubert
Klaus
K
D000626:Aminopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D018914:Cells, Immobilized / Q000201:enzymology; D045604:Extracellular Fluid / Q000201:enzymology*; D007424:Intracellular Fluid / Q000201:enzymology*; D018551:Solanum lycopersicum / Q000166:cytology* / Q000201:enzymology*
D000626:Aminopeptidases