Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.     

Human erythrocyte glutathione reductase: pH dependence of kinetic parameters

Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH, 2',3'-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2',3'-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's for ATP-ribose and 2',5'-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2'-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467'-Glu-472' ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.     

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.     

Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism

Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.     

Dynamics of carbon monoxide binding with neuronal nitric oxide synthase.

The dynamics of CO rebinding with neuronal NO synthase (nNOS) following laser flash photolysis have been investigated from 293 to 77 K in the absence and presence of its substrate L-arginine. The distribution functions of the rate parameters P(k) and of the activation enthalpy P(H) were determined using the maximum entropy method. In a fluid solvent near room temperature, bimolecular rebinding is biphasic, as previously reported by several groups. However, measurement of the rotational correlation time shows that the apparent biphasic rebinding is not relevant to the genuine dynamics of NOS. In addition to native dimeric nNOS, another species (possibly aggregated or partially unfolded conformation) with different hydrodynamic characteristics is responsible for the faster rebinding process. In a rigid environment at low temperature, the geminate internal rebinding is not affected by the presence of the nonnative species. nNOS exhibits a bimodal distribution of CO activation enthalpy with P(H) consisting of two distinct bands with temperature-dependent amplitudes down to 77 K. The similarity of these findings with those recently reported for cytochromes P-450 suggests a common hierarchical organization of conformational substates, with a splitting of each conformational substate into a doublet. Thus, thiolate-coordinated heme proteins are in clear contrast to histidine-coordinated oxygen-transport heme proteins. The present results with nNOS provide additional support to previous arguments incriminating the thiolate ligand as responsible for the splitting of conformational substates.     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.

The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.     

Calcium-deficient calmodulin binding and activation of neuronal and inducible nitric oxide synthases

The nitric oxide synthase (NOS) enzymes are bound and activated by the Ca(2+)-binding protein, calmodulin (CaM). We have utilized CaM mutants deficient in binding Ca(2+) with mutations in the N-lobe (CaM(12)), the C-lobe (CaM(34)), or both lobes of CaM (CaM(1234)) to determine their effect on the binding and activation of the Ca(2+)-dependent neuronal (nNOS) and Ca(2+)-independent inducible NOS (iNOS) isoforms. Four different kinetic assays were employed to monitor the effect of these CaM mutants on electron transfer rates in NOS. Protein-protein interactions between CaM and NOS were studied using steady-state fluorescence and spectropolarimetry to monitor the binding of these CaM mutants to nNOS and iNOS CaM-binding domain peptides. The CaM mutants were unable to activate nNOS, however, our CD results show that the C-terminal lobe of CaM is capable of binding to nNOS peptide in the presence of Ca(2+). Our results prove for the first time without the use of chelators that apo-CaM is capable of binding to iNOS peptides and holoenzymes.     

Regulation of RYR1 activity by Ca(2+) and calmodulin

The skeletal muscle calcium release channel (RYR1) is a Ca(2+)-binding protein that is regulated by another Ca(2+)-binding protein, calmodulin. The functional consequences of calmodulin's interaction with RYR1 are dependent on Ca(2+) concentration. At nanomolar Ca(2+) concentrations, calmodulin is an activator, but at micromolar Ca(2+) concentrations, calmodulin is an inhibitor of RYR1. This raises the question of whether the Ca(2+)-dependent effects of calmodulin on RYR1 function are due to Ca(2+) binding to calmodulin, RYR1, or both. To distinguish the effects of Ca(2+) binding to calmodulin from those of Ca(2+) binding to RYR1, a mutant calmodulin that cannot bind Ca(2+) was used to evaluate the effects of Ca(2+)-free calmodulin on Ca(2+)-bound RYR1. We demonstrate that Ca(2+)-free calmodulin enhances the affinity of RYR1 for Ca(2+) while Ca(2+) binding to calmodulin converts calmodulin from an activator to an inhibitor. Furthermore, Ca(2+) binding to RYR1 enhances its affinity for both Ca(2+)-free and Ca(2+)-bound calmodulin.     

Rate, affinity and calcium dependence of nitric oxide synthase isoform binding to the primary physiological regulator calmodulin

Using interferometry-based biosensors the binding and release of endothelial and neuronal nitric oxide synthase (eNOS and nNOS) from calmodulin (CaM) was measured. In both isoforms, binding to CaM is diffusion limited and within approximately three orders of magnitude of the Smoluchowski limit imposed by orientation-independent collisions. This suggests that the orientation of CaM is facilitated by the charge arrays on the CaM-binding site and the complementary surface on CaM. Protein kinase C phosphorylation of eNOS T495, adjacent to the CaM-binding site, abolishes or greatly slows CaM binding. Kinases which increase the activity of eNOS did not stimulate the binding of CaM, which is already diffusion limited. The coupling of Ca(2+) binding and CaM/NOS binding equilibria links the affinity of CaM for NOS to the Ca(2+) dependence of CaM binding. Hence, changes in the Ca(2+) sensitivity of CaM binding always imply changes in the NOS-CaM affinity. It is possible, however, that in some regimes binding and activation are not synonymous, so that Ca(2+) sensitivity need not be tightly linked to CaM sensitivity of activation. This study is being extended using mutants to probe the roles of individual structural elements in binding and release.     

Interaction between inducible nitric oxide synthase and calmodulin in Ca2+-free and -bound forms

We have obtained the first direct electrochemistry of full-length inducible nitric oxide synthase (iNOS) by entrapping the enzyme in polyethylenimine (PEI) film. The interaction between iNOS and calmodulin (CaM) was then studied, which revealed an enhanced electron-transfer reactivity of the enzyme facilitated by CaM. It was also found that interflavin electron transfer of iNOS could be activated by the binding of Ca2+-bound CaM. The formal potentials (E degrees ') of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were determined to be -470 and -284 mV vs SCE at pH 7, respectively. The effect of Ca2+ on the interaction between iNOS and CaM has been examined as well. CaM bound with adequate Ca2+ was shown to have a better capability to enhance the electron-transfer reactions within iNOS.     

Effects of Ca(2+) and calmodulin on the motile activity of characean myosin in vitro

It is well known that the cytoplasmic streaming of characean cells is readily inhibited by Ca(2+). However, neither the actin-activated MgATPase nor the in vitro motile activity of purified characean myosin were inhibited by Ca(2+). Recently, amino acid sequence of characean myosin was determined in our laboratory and the sequence revealed that characean myosin contains six calmodulin binding sites in the neck region. We also detected calmodulin in quickly prepared characean myosin fraction. It is, therefore, possible that the insensitivity of characean myosin to Ca(2+) is due to the dissociation of some calmodulin molecules from the neck region during the course of protein purification. To determine strictly the Ca(2+) sensitivity of characean myosin, we intentionally used crude preparation of characean myosin to reduce the possibility of calmodulin dissociation and examined the motile activity of characean myosin in vitro in the presence of excess characean calmodulin. We could not observe any drastic inhibition of characean myosin activity by Ca(2+). The results suggest that the brief cessation of cytoplasmic streaming is not caused by the direct inhibition of myosin activity by Ca(2+).     

FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.     

Differential binding of calmodulin domains to constitutive and inducible nitric oxide synthase enzymes

Calmodulin (CaM) is a Ca2+ signal transducing protein that binds and activates many cellular enzymes with physiological relevance, including the mammalian nitric oxide synthase (NOS) isozymes: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). The mechanism of CaM binding and activation to the iNOS enzyme is poorly understood in part due to the strength of the bound complex and the difficulty of assessing the role played by regions outside of the CaM-binding domain. To further elucidate these processes, we have developed the methodology to investigate CaM binding to the iNOS holoenzyme and generate CaM mutant proteins selectively labeled with fluorescent dyes at specific residues in the N-terminal lobe, C-terminal lobe, or linker region of the protein. In the present study, an iNOS CaM coexpression system allowed for the investigation of CaM binding to the holoenzyme; three different mutant CaM proteins with cysteine substitutions at residues T34 (N-domain), K75 (central linker), and T110 (C-domain) were fluorescently labeled with acrylodan or Alexa Fluor 546 C5-maleimide. These proteins were used to investigate the differential association of each region of CaM with the three NOS isoforms. We have also N-terminally labeled an iNOS CaM-binding domain peptide with dabsyl chloride in order to perform FRET studies between Alexa-labeled residues in the N- and C-terminal domains of CaM to determine CaM's orientation when associated to iNOS. Our FRET results show that CaM binds to the iNOS CaM-binding domain in an antiparallel orientation. Our steady-state fluorescence and circular dichroism studies show that both the N- and C-terminal EF hand pairs of CaM bind to the CaM-binding domain peptide of iNOS in a Ca2+-independent manner; however, only the C-terminal domain showed large Ca2+-dependent conformational changes when associated with the target sequence. Steady-state fluorescence showed that Alexa-labeled CaM proteins are capable of binding to holo-iNOS coexpressed with nCaM, but this complex is a transient species and can be displaced with the addition of excess CaM. Our results show that CaM does not bind to iNOS in a sequential manner as previously proposed for the nNOS enzyme. This investigation provides additional insight into why iNOS remains active even under basal levels of Ca2+ in the cell.     

Phosphorylation within an autoinhibitory domain in endothelial nitric oxide synthase reduces the Ca(2+) concentrations required for calmodulin to bind and activate the enzyme

We have investigated the effects of phosphorylation at Ser-617 and Ser-635 within an autoinhibitory domain (residues 595-639) in bovine endothelial nitric oxide synthase on enzyme activity and the Ca (2+) dependencies for calmodulin binding and enzyme activation. A phosphomimetic S617D substitution doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and enzyme activation from the wild-type values of 180 +/- 2 and 397 +/- 23 nM to values of 109 +/- 2 and 258 +/- 11 nM, respectively. Deletion of the autoinhibitory domain also doubles the maximum calmodulin-dependent enzyme activity and decreases the EC 50(Ca (2+)) values for calmodulin binding and calmodulin-dependent enzyme activation to 65 +/- 4 and 118 +/- 4 nM, respectively. An S635D substitution has little or no effect on enzyme activity or EC 50(Ca (2+)) values, either alone or when combined with the S617D substitution. These results suggest that phosphorylation at Ser-617 partially reverses suppression by the autoinhibitory domain. Associated effects on the EC 50(Ca (2+)) values and maximum calmodulin-dependent enzyme activity are predicted to contribute equally to phosphorylation-dependent enhancement of NO production during a typical agonist-evoked Ca (2+) transient, while the reduction in EC 50(Ca (2+)) values is predicted to be the major contributor to enhancement at resting free Ca (2+) concentrations.     

Inhibitory effect of organotin compounds on rat neuronal nitric oxide synthase through interaction with calmodulin

Organotin compounds, triphenyltin (TPT), tributyltin, dibutyltin, and monobutyltin (MBT), showed potent inhibitory effects on both L-arginine oxidation to nitric oxide and L-citrulline, and cytochrome c reduction catalyzed by recombinant rat neuronal nitric oxide synthase (nNOS). The two inhibitory effects were almost parallel. MBT and TPT showed the highest inhibitory effects, followed by tributyltin and dibutyltin; TPT and MBT showed inhibition constant (IC(50)) values of around 10microM. Cytochrome c reduction activity was markedly decreased by removal of calmodulin (CaM) from the complete mixture, and the decrease was similar to the extent of inhibition by TPT and MBT. The inhibitory effect of MBT on the cytochrome c reducing activity was rapidly attenuated upon dilution of the inhibitor, and addition of a high concentration of CaM reactivated the cytochrome c reduction activity inhibited by MBT. However, other cofactors such as FAD, FMN or tetrahydrobiopterin had no such ability. The inhibitory effect of organotin compounds (100microM) on L-arginine oxidation of nNOS almost vanished when the amount of CaM was sufficiently increased (150-300microM). It was confirmed by CaM-agarose column chromatography that the dissociation of nNOS-CaM complex was induced by organotin compounds. These results indicate that organotin compounds disturb the interaction between CaM and nNOS, thereby inhibiting electron transfer from the reductase domain to cytochrome c and the oxygenase domain.     

Ca(2+) and Zn(2+) binding properties of peptide substrates of vertebrate collagenase, MMP-1.

To understand the role of Ca(2+) in vertebrate in the structure and action of collagenase, we have examined peptides that interact with recombinant human fibroblast collagenase for their affinities towards Ca(2+) and Zn(2+) in a non-polar solvent. Two of the peptides, GPQGIAGQ and GNVGLAGA, had sequences in collagen which are, respectively, cleaved and not cleaved by collagenase. A third peptide, PSYFLNAG, had a collagenase-cleaved sequence in ovostatin, a globular protein substrate. Peptides TVGCEECTV and CLPREPGL were derived from TIMP-1; the former competitively inhibits collagenase while the latter does not. The relative rates of hydrolysis of the peptides by collagenase had the order GPQGIAGQ>PSYFLNAG>GNVGLAGA. Circular dichroism spectral data in trifluoroethanol showed that while the TIMP control peptide, CLPREPGL, bound only Zn(2+), the other four peptides bound both Ca(2+) and Zn(2+) with definite stoichiometries. Ca(2+) could displace Zn(2+) in the substrate peptides while Zn(2+) displaced Ca(2+) in the TIMP peptide. GPQGIAGQ, PSYFLNAG and TVGCEECTV formed peptide:Ca(2+):Zn(2+) ternary complexes. Our results suggest that both collagen and globular protein substrates of collagenase may bind Ca(2+) and Zn(2+) in the enzyme's active site. This, in turn, may account for the known importance of the non-catalytic Ca(2+) and Zn(2+) in collagenase activity.     

Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release.

We examined the possibility that Ca2+ released from intracellular stores could activate K+ currents underlying the afterhyperpolarization (AHP) in neurons. In neurons of the dorsal motor nucleus of the vagus, the current underlying the AHP had two components: a rapidly decaying component that was maximal following the action potential (GkCa,1) and a slower component that had a distinct rising phase (GkCa,2). Both components required influx of extracellular Ca2+ for their activation, and neither was blocked by extracellular TEA (10 mM). GkCa,1 was selectively blocked by apamin, whereas GkCa,2 was selectively reduced by noradrenaline. The time course of GkCa,2 was markedly temperature sensitive. GkCa,2 was selectively blocked by application of ryanodine or sodium dantrolene, or by loading cells with ruthenium red. These results suggest that influx of Ca2+ directly gates one class of K+ channels and leads to release of Ca2+ from intracellular stores, which activates a different class of K+ channel.