Identification and determination of the intra- and extracellular aminopeptidase activity by synthetic L-Ala-, L-Tyr-, and L-Phe-beta-napthylamide.
A simple, rapid and straightforward procedure for identification and determination of intracellular and extracellular activity of aminopeptidases employing synthetic substrates beta-naphtylamides of L-Ala, L-Phe, and L-Tyr was used. Poppy cells (Papaver somniferum L.) permeabilized by Tween 80 were immobilized via crosslinking by glutaraldehyde. Glutaraldehyde immobilized poppy cells lost their viability and demonstrated significantly lower aminopeptidase activities than untreated control cells probably due to a damage to the enzyme active centre. Poppy cells immobilized by pectate and alginate have retained high activity of studied aminopeptidases. The culture medium (without cells) used for the identification and determination of extracellular enzyme activities retained 20-21%, whereas intracellular activities were estimated to be 79-80% of total enzyme activity. Thus the intracellular specific activity was 1.00-1.07 higher.
Stano
J
J
Garden of Medicinal Plants, Comenius University, Bratislava, Slovak Republic.
Siekel
P
P
Micieta
K
K
Blanárikova
V
V
Korenová
M
M
Bergerová
E
E
Nemec
P
P
D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D000464:Alginates / Q000737:chemistry; D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002470:Cell Survival; D002478:Cells, Cultured; D005110:Extracellular Space / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D042541:Intracellular Space / Q000201:enzymology; D010207:Papaver / Q000201:enzymology*; D010368:Pectins / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D011136:Polysorbates; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
D000464:Alginates; D000577:Amides; C538766:L-Phe-beta-napthylamide; C538767:L-ala-beta-napthylamide; C538768:L-tyr-beta-napthylamide; D011136:Polysorbates; D014443:Tyrosine; D010649:Phenylalanine; D010368:Pectins; D000626:Aminopeptidases; D005947:Glucose; D000409:Alanine; D005976:Glutaral; C003181:polygalacturonic acid