ABSTRACT
Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3' sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
Subject(s)
Calcium/metabolism , Cellulase/metabolism , Clostridium/enzymology , Amino Acid Sequence , Cellulase/genetics , Clostridium/genetics , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , Plasmids , Sequence Homology, Nucleic AcidABSTRACT
Ingestion of 750 ml cider inhibited plasma fibrinolytic activity: unfermented apple juice appeared to have some inhibitory effect, but this did not reach statistical significance. Cider inhibited urokinase-induced clot lysis in a concentration-dependent manner; the inhibitory activity was heat-stable and non-dialysable. The fibrinolytic activities of plasmin, urokinase and, more markedly, tissue activator on fibrin plates were inhibited by cider in a concentration-dependent manner. The amidolytic activity of plasmin was also inhibited in the presence of cider.