TLCKTosyl Lysyl Chloromethyl Ketone
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Trypsin activity was inhibited by 63% for females and 75% for males, using TLCK as an inhibitor; the serine protease activity was inhibited by 91% for females and 87% for males using PMSF, and 85% for females and
With the use of OVO (line 2), only one band (28.3 kDa) was inhibited, using SBT1 and Phen (lines 3 and 6 respectively) the same two bands (45.8 and 28.5 kDa) were inhibited, while using the TPCK (line 5), bands of 45.8 and 94.0 kDa were inhibited; on the other hand, with EDTA and TLCK (lines 6 and 7 respectively) only one band was inhibited (28.5 kDa); finally, the PMSF (line 4) inhibited the total of the bands with proteolytic activity (Fig.
Proteases in which N-p-tosyl-L-lysine chloromethyl ketone (TLCK) and phenylmethanesulphonyl fluoride (PMSF) diminished their activity were designated trypsins.
Three isotrypsins were identified in shrimp larvae by inhibition of TLCK and PMSF, corresponding to trypsins A, B, and C (Sainz et al., 2004).
MMVECs were seeded onto 96-well cell culture plates at an initial density of 5 x [10.sup.3] cells/well (100 [micro]L) in DMEM with 5% FBS in the presence or absence of MCGs, TLCK, and TPCK.
Inserts were placed on a 24-well plate containing 800 mL DMEM and 2% FBS in the presence or absence of MCGs, TLCK, and TPCK.
Abbreviations of Inhibitors and Activators of Proteases AEBSF: 4-(2-Aminoethyl) benzenesulfonyl fluoride DIFP: Diisopropyl fluorophosphates DTT: Dithiothreitol EDTA: Ethylenediaminetetraacetic acid E64: Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane IA: Iodoacetamide NEM: N-ethylmaleimide PHMB: p-Hydroxymercuribenzoate PMSF: Phenylmethylsulfonylfluoride TLCK: Tosyl-lysyl-chloromethylketone TPCK: 1-Chloro-3-tosylamido-4-phenyl-2-butanone Z-FA-FMK: Benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone.
Los efectos del PMSF y TLCK evidenciarian la presencia de los aminoacidos serina e histidina y/o cisteina como residuos fundamentales en la estructura catalitica.
Pepstatin A (1 mmol [L.sup.-1]) was used as an inhibitor of acid proteases from stomach and alkaline protease activity inhibition in pyloric caeca sections were performed using the following inhibitors: 250 mmol [L.sup.-1] soybean trypsin inhibitor (SBT1), 10 mol [L.sup.-1] N-tosyl-L-phenylchloromethyl ketone (TPCK), 100 mmol [L.sup.-1] phenylmethylsulfonyl fluoride (PMSF), 10 mmol [L.sup.-1] Na-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), 10 mmol [L.sup.-1] 1, 10-Phenanthroline (Phen) and 250 mmol [L.sup.-1] Type II-Turkey egg Ovomucoid (Ovo).
The effect of inhibitors [10 [micro]mol [1.sup.-1] soybean trypsin inhibitor (SBTI), 10 mmol [1.sup.-1] benzamidine, 1 mmol [1.sup.-1] phenyl methyl sulfonyl fluoride (PMSF), 10 [micro]mol [1.sup.-1] aprotinin, 1 mmol [1.sup.-1] N[alpha]-p-tosyl-L-lysine chloromethyl ketone (TLCK), 1 mmol [1.sup.-1] ethylene-diaminetetraacetic acid (EDTA), 0.1 mmol [1.sup.-1] leupectin, 1 mmol [1.sup.-1] NEM)] was evaluated by incubating the isolated enzymes with inhibitors for 30 min before measuring trypsin activity using BApNA as describe above.