Z leu leu glu amc

The substrates Suc-Leu-Leu-Val-Tyr-7-Amino-4-methylcoumarin (AMC), Z-Leu-Ser-Thr-Arg-AMC, Z-Leu-Leu-Glu-AMC for assaying the chymotrypsin-like (ChT-L), trypsin-like (T-L), and peptidyl glutamyl-peptide hydrolyzing (PGPH) proteasomal activities were purchased from Sigma-Aldrich S.r.L. (Milano, Italy). Z-Gly-Pro-Ala-Leu-Ala-MCA to measure BrAAP activity was from Biomatik (Cambridge, Ontario). Aminopeptidase N (EC 3.4.11.2) was purified from pig kidney [39 ]. Cathepsin B and cathepsin L substrates (Z-Arg-Arg-AMC and Z-Phe-Arg-7-amino-4-trifluoromethyl-coumarin (AFC) respectively), cathepsins inhibitors (CA074Me and N-(1-Naphthalenylsulfonyl)-Ile-Trp-aldehyde), and monodansylcadaverine (MDC) were obtained from Sigma-Aldrich S.r.L. (Milano, Italy). Media and chemicals used for cell cultures were purchased from Enzo Life Sciences, Inc. The membranes for western blot analyses were purchased from Millipore (Milan, Italy). Proteins immobilized on polyvinylidene fluoride membranes were detected with the enhanced chemiluminescence (ECL) technique (Amersham Pharmacia Biotech, Milan, Italy) on a ChemiDoc MP, Chemiluminescence system (Biorad, Italy).

5‐(4ʹ‐hydroxyphenyl)‐γ‐valerolactone (C1), 5‐(3ʹ,4ʹ‐dihydroxyphenyl)‐γ‐valerolactone (C2) and 5‐(3ʹ‐hydroxyphenyl)‐γ‐valerolactone‐4ʹ‐sulfate (C3) (Figure1) were synthesized and kindly provided by Prof. C. Curti (University of Parma, Italy)^.[14, ^15] The substrates Suc‐Leu‐Leu‐Val‐Tyr‐AMC, Z‐Leu‐Ser‐Thr‐Arg‐AMC, Z‐Leu‐Leu‐Glu‐AMC for assaying the chymotrypsin‐like (ChT‐L), trypsin‐like (T‐L), and peptidyl glutamyl‐peptide hydrolyzing (PGPH) activities of the proteasomal complex were purchased from Sigma‐Aldrich S.r.L. (Milano, Italy). The substrate Z‐Gly‐Pro‐Ala‐Leu‐Ala‐MCA to test the branched chain amino acids preferring (BrAAP) activity was obtained from Biomatik (Cambridge, Ontario). Aminopeptidase N (EC 3.4.11.2) for the coupled assay utilized to detect BrAAP activity was purified from pig kidney as reported elsewhere.^[16] Membranes for western blot analyses were purchased from Millipore (Milan, Italy). Proteins immobilized on films were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Milan, Italy). All chemicals and solvents were of the highest analytical grade available.