mx Abbreviations: BAPNA, M[alpha]-benzoyl-DL-arginine-p-nitroanilide; mUA, milliunits of activity (in absorbance); PMSF, phenylmethanesulphonyl fluoride; SAPNA, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TLCK
, N-p-tosyl-L-lysine chloromethyl ketone.
SBTI (soybean trypsin inhibitor), TPCK (N-tosyl-L-phenyl -chloromethyl ketone), PMSF (phenylmethylsulfonyl fluoride), TLCK
(N[alpha]-tosyl-L-lysine chloromethyl ketone hydrochloride), Phen (1.
Cells were washed with PBS and further incubated in DMEM with 1% FBS in the presence or absence of MCGs, TLCK, and TPCK.
After solidification at 37[degrees]C for 30 min, MMVECs resuspended in DMEM with 1% FBS in the presence or absence of MCGs, TLCK, and TPCK were added onto the surface of the Matrigel (5 x [10.
However, these effects were totally abolished when TLCK or TPCK was added, resulting in an even lower proliferation rate than in MMVECs alone (P<0.
More importantly, the effects of MCGs were significantly suppressed by adding TLCK and TPCK, resulting in an even lower migration and capillary-like tube formation than in MMVECs alone (P<0.
Both TLCK and TPCK abolished the effects of MCGs on Ang-1 and Ang-2 mRNA expression, without affecting Tie-2 mRNA expression (Figure 3D-F).
This suppressive effect was removed by adding either TLCK or TPCK (P<0.
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.
Agente quimico Actividad especifica (%) 2,5 mM 5 mM 10 mM 20 mM Control 100% DTT 90,3 73,0 65,0 62,0 2-Mercaptoetanol 89,5 81,7 72,4 59,4 Iodoacetato 92,6 81,0 63,7 55,3 TLCK 88,0 76,1 58,1 49,5 PMSF 68,1 53,0 42,4 39,9 EDTA 70,8 52,4 35,8 32,7 Acido aspartico 98,0 77,5 64,4 62,1 Acido glutamico 90,9 69,2 51,3 47,7 Cisteina 80,3 54,9 41,6 31,6 Glutation 78,1 48,9 36,4 30,0 Figura 2.
Inhibitors of serine proteases like SBTI and aprotinin strongly inhibited the activity of all enzymes, as did the trypsin inhibitor TLCK (Table 2).
Also, since TLCK and PMSF react with the active site His and Ser residues, respectively, the strongest effect of these inhibitors on PaTS could be related to the higher catalytic efficiency of this form of the enzyme (see below).