PubMed · 872389
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R L Stux, R H Emmel, H L Kahn. 1977. Improved spectrophotometer evaluated.. https://pubmed.ncbi.nlm.nih.gov/872389/
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In calcium signaling pathways regulated by the EF-hand Ca2+ binding motif, proper regulation requires that the equilibrium and kinetics of Ca2+ binding to the EF-hand chelation loop be precisely optimized for each physiological application. Studies of small-molecule organic chelators have shown that metal binding parameters can be tuned both by the nature of the coordinating ligands and by the structural framework to which these ligands are attached. By analogy, the present study tests the relative importance of (i) coordinating side chains and (ii) backbone torsion angle constraints to the tuning of an EF-hand-like Ca2+ chelation loop. A series of engineered chelation loops are generated by modifying Ca2+ binding site of the Escherichia coli galactose binding protein. The resulting loops, each containing an altered coordinating side chain or a Gly substitution, are compared with respect to their metal binding affinities, specificities, and dissociation kinetics. The Gly variants examined include substitutions which eliminate or introduce a Gly at each of the nine chelation loop positions. The results reveal that Gly is not tolerated at loop positions 1, 3, 5, or 8 or at the external coordinating position, where the removal of a key coordinating or hydrophobic side chain destabilizes the protein. In contrast, Gly residues at loop positions 2, 4, 6, and 7, none of which is required for side chain coordination, have little effect on Ca2+ affinity and the ability to discriminate between cations of different size and charge. Kinetic measurements show that some of these Gly residues measurably alter the rates of metal ion association and dissociation, but in each case the two rates are changed by approximately the same factor so that the effects on equilibrium are minor. Overall, Gly residues yield surprisingly small effects at loop positions 2, 4, 6, and 7, especially when compared to the larger equilibrium and kinetic effects observed for coordinating side chain substitutions. It follows that the conserved Gly at position 6 is not required for Ca2+ binding and that constraints on the backbone torsion angles at the non-coordinating side chain positions 2, 4, 6, and 7 play a relatively minor role in tuning metal binding parameters. Instead, specific coordinating side chains optimize the metal binding parameters of the GBP chelation loop for its protein context and biological application.
The effect of human neutrophil elastase (HNE) on human factor V (F.V) or alpha-thrombin-activated human factor V (F.Va) was studied in vitro by prothrombinase assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and NH2-terminal sequence analysis. Incubation of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in a time-dependent increase in its cofactor activity. In contrast, treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted only in a time-dependent decrease in its cofactor activity. Under the conditions of these experiments, the maximum extent of F.V activation accomplished by incubation with HNE was approximately 65% to 70% of that observed with alpha-thrombin in presence of Ca2+. The extent of both the HNE-dependent enhancement in F.V cofactor activity and the HNE-dependent decrease in F.Va cofactor activity was not influenced by the addition of phosphatidylcholine/phosphatidylserine (PCPS) vesicles (50 micromol/L). The HNE-derived cleavage products of F.V, which correlated with increased cofactor activity, as demonstrated by SDS-PAGE under reducing conditions, were different from those generated using alpha-thrombin. Treatment of F.V (600 nmol/L) with HNE (2 nmol/L) in the presence of Ca2+ resulted in the production of three closely spaced doublets of: 99/97, 89/87, and 76/74 kD whose appearance over time correlated well with the increased cofactor activity as judged by densitometry. Treatment of F.Va (600 nmol/L) with HNE (60 nmol/L) in the presence of Ca2+ resulted in the cleavage of both the 96 kD heavy chain and the 74/72 kD light chain into products of: 56, 53, 35, 28, 22, and 12 kD. Although densitometry indicated that both the heavy and light chains of F.Va were hydrolyzed by HNE, cleavage of the 96 kD heavy chain was more extensive during the time period (10 to 30 minutes) of the greatest loss of F.Va cofactor activity. NH2-terminal sequence analysis of F.V treated with HNE indicated cleavage at Ile819 and Ile1484 under conditions during which the procofactor expressed enhanced cofactor activity in the prothrombinase complex. NH2-terminal sequence analysis of F.Va treated with HNE indicated cleavage at Ala341, Ile508, and Thr1767 under conditions, which the cofactor became inactivated, as measured by prothrombinase activity. The activation and inactivation cleavage sites are close to those cleaved by the physiological activator and inactivator of F.V and F.Va, namely alpha-thrombin (Arg709 and Arg1545) and Activated Protein C (APC) (Arg306 and Arg506), respectively. These results indicate that HNE can generate proteolytic products of F.V, which initially express significantly enhanced procoagulant cofactor activity similar to that observed following activation with alpha-thrombin. In contrast, HNE treatment of F.Va resulted only in the loss of its cofactor activity, but again, this is similar to that observed following inactivation by APC.
Modulation of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities by delta-opioid receptor specific agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) was investigated in neuroblastoma x glioma hybrid NG 108-15 cells. DPDPE activated PKC in a dose-dependent manner, with the maximal response at 5 min. The DPDPE-stimulated PKC activation could be blocked by naltrindole. The activation of PKC by DPDPE was dependent on Ca2+ and was inhibited by chelerythrine chloride (10 microM), but not by H89 (1 microM). Pretreatment of NG 108-15 cells with pertussis toxin (100 ng/ml for 24 h) completely abolished DPDPE-stimulated PKC activation. In contrast to the result from the acute treatment with DPDPE, which had no significant effect on PKA activity, chronic treatment of DPDPE (1 microM for 24 h) increased PKA activity, but reduced the basal activity of PKC. These results demonstrated that DPDPE differentially modulated PKC and PKA activities via a receptor-mediated, PTX sensitive pathway.