4) containing 1 mM EDTA, 1 mM dithiothreitol, 5 mg/L phenylmethylsulfonyl fluoride, 5 mg/L aprotinin, 5 mg/L chymostatin
, and 5 mg/L pepstatin added phosphatase inhibitor, sodium orthovanadate to a final concentration of 2 mM.
Effect of protease inhibitors on endogenous or exogenous [beta]-galactosidase in human fibroblasts Inhibitor Protease [beta]- [beta]- Galactosidase Galactosidase (endogenous) * (exogenous) ** None 100 100 E-64 Thiol 430 229 (Cysteine) *** Leupeptin Thiol 460 222 (Cysteine) *** Antipain Thiol 490 224 (Cysteine) *** Chymostatin
Thiol 520 188 (Cysteine) *** Elastatinal Elastase 60 103 Pepstatin Pepsin 100 97 Phosphoramidon Metallopeptidase 110 88 Bestatin Aminopeptidase 110 90 * Relative enzyme activity in human fibroblasts with cathepsin A deficiency (galactosialidosis).
Powdered biomass was gently suspended in 5 mL of extraction buffer (10mM Tris HCL pH8; 1mM EDTA, 2% (w/v) polivinilpolipirolidona PVPP) containing protease inhibitors (1ug/mL chymostatin
, 1 [micro]g/mL aprotinin, 1ug/mL leupeptin and 2mM PMSF), and centrifuged at 8000 rpm for 30 min at 4 [degrees]C; then, supernatant was collected.
This theory is reinforced by the fact that the replacement of tyrosine-alanine with asparagine-proline at that residue in the sequence of guanylin turns it resistant to protease (Carpick and Gariepy, 1993) and the blockade of guanylin degradation by another chymotrypsin inhibitor named chymostatin
(Santos-Neto et al.
1]) phenylmethylsulfonyl fluoride (PMSF), antipain, leupeptin, pepstatin A, and chymostatin
As assayed after a 10-min exposure, only chymostatin and AEBSF were effective, at 18% and 7% X-cells, respectively (vs.
Transformation was also greatly reduced by preheating of hemolymph and blocked by the serine protease inhibitors AEBSF and chymostatin.
5, containing 2 mmol/L dithiothreitol, 2 mmol/L EDTA, 1 mmol/L EGTA, 5 g/ml each of leupeptin, aprotinin, pepstatin A and chymostatin
, 50 mmol/L potassium fluoride, 50 mmol/L okadaic acid, 5 mmol/L sodium pyrophosphate).
13 respectively, relative to chymostatin
having IC50 value of 8.
Diquat (dibromide monohydrate, West Chester, PA), D-mannitol, HEPES, dithiothreitol, EDTA, phenylmethyl-sulfonyl fluoride, aprotinin, chymostatin
, pepstatin A, Triton X-100, L-ornithine, pyridoxal phosphate, [alpha]-ketoglutarate, o-aminobenzaldehyde, o-phthaldialdehyde, L-arginine, L-citrulline (Sigma, Buchs, Italy) were procured from Sigma (St.
was used as standard inhibitor in this assay (IC50 = 5.
02, respectively whereas chymostatin
is used as positive control (IC50 = 8.