Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A soluble 80-kDa endopeptidase has been isolated from Trypanosoma brucei brucei. The enzyme, which has a pI 5.1, is optimally active at about pH 8.2 and has apparent pKa values of 6.0 and greater than or equal to 10. It is inhibited by the serine protease inhibitor diisopropylfluorophosphate and by the serine protease mechanism-based inhibitor 3,4-dichloroisocoumarin. Unexpectedly, the enzyme is inhibited by the cysteine protease inhibitor benzyloxycarbonyl-Leu-Lys-CHN2 but not by the related diazomethane, butoxycarbonyl-Val-Leu-Gly-Lys-CHN2, nor by other cysteine protease specific compounds. Specificity studies with a variety of amidomethylcoumaryl (AMC) derivatives of small peptides show that the enzyme has a highly restricted trypsin-like specificity. The best substrate, based on the magnitude of kcat/Km, was benzyloxycarbonyl-Arg-Arg-AMC; other good substrates were benzyloxycarbonyl-Phe-Arg-AMC, benzoyl-Arg-AMC, and compounds with Arg at P1 and Ala or Gly at P2. The hydrolysis of most substrates obeyed classical Michaelis-Menton kinetics but several exhibited pronounced substrate inhibition. The enzyme did not activate plasminogen nor decrease blood clotting time; it was inhibited by aprotinin but not by chicken ovomucoid. We conclude that the enzyme is a trypsin-like serine endopeptidase with unusually restricted subsite specificities.
 ...
PMID:Characterization of an endopeptidase of Trypanosoma brucei brucei. 173 36

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsin-like activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin-like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyl- and radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3, 4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.
 ...
PMID:Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. 931 91