Preferential ligand binding to multi-state acceptor systems: comparisons of the calcium-binding and dimerization characteristics of prothrombin and fragment 1.

Consideration is given to the interactions of a ligand with self-associating acceptor systems for which preferential binding is an ambiguous term in that ligand-mediated self-association does not necessarily imply a greater binding constant for polymeric acceptor--even in instances where binding sites are preserved in the self-association process. This dilemma is shown to arise in situations involving the binding of ligand to monomeric and polymeric forms of an acceptor that also coexist in equilibrium with inactive isomeric states. For example, the ten-fold increase in the measured dimerization constant for prothrombin Fragment 1 in the presence of a saturating concentration of Ca2+ ion may well reflect the existence of a 12% greater binding constant for the interaction of metal ion with dimeric acceptor. However, that result, as well as the detailed form of the sigmoidal binding curve, are also reasonably described by another extreme model in which the monomeric and dimeric forms of the acceptor possess equal affinities for Ca2+ ion. Likewise, the fact that the same experimental dimerization constant applies to prothrombin and its Ca(2+)-saturated complex does not preclude the possibility that the active form of dimeric zymogen exhibits a 12% greater affinity for metal ion. Numerical simulations have established that characterization of the dimerization behaviour as a function of free ligand concentration should allow greater discrimination between such models of the interplay between calcium binding and self-association of prothrombin and Fragment 1. Finally, by illustrating the likelihood that the disparity in self-association behaviour of prothrombin and Fragment 1 merely reflects minor differences in the relative magnitudes of isomerization constants and/or binding constants for monomeric and dimeric states of the two acceptors, the present investigation serves to allay concern about the validity of employing the proteolytic fragment as a model of the intact zymogen.     

The effect of divalent metal ions on the electrophoretic mobility of bovine prothrombin and bovine prothrombin fragment 1

Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.     

Dye binding probes of lipid-binding structures. An investigation of 2-p-toluidinylnaphthylene-6-sulfonate binding to human and bovine prothrombin and fragment 1 in the presence and absence of calcium and magnesium ions

TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.     

Evidence for self-association of prothrombin fragment 1 in the absence of calcium ions. Implications for the interpretation of cooperativity of calcium binding

Sedimentation equilibrium studies have demonstrated that prothrombin fragment 1 from either human or bovine plasma reversibly dimerizes in the absence of Ca2+ with an equilibrium constant of 1,000 M-1. In the presence of 10 mM Ca2+ this association constant increased to 10,000 M-1. A model for preferential binding of Ca2+ to the pre-existing dimer has been found capable of accounting quantitatively for the cooperative Ca2+ binding to this prothrombin fragment, and for the dependence of its sedimentation coefficient on protein concentration in the presence and absence of metal ion. Sedimentation equilibrium studies of intact bovine and human prothrombins have confirmed previous reports that these prothrombins dimerize. For both prothrombins the association constant is 10,000 M-1, both in the absence and presence of Ca2+.     

Selective affinity chromatography with calmodulin fragments coupled to sepharose

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

The effect of a lipophilic antibiotic, ionophore A23187, on the purified Ca2+-ATPase from sarcoplasmic reticulum was investigated. When the enzyme was pretreated with A23187 in the presence and absence of Ca2+, the Ca2+-dependent ATPase activity was inhibited almost completely, but the activity of the contaminating Mg2+-ATPase was unaffected. The steady state level of the phosphoenzyme (EP) from ATP or Pi was not substantially altered. When the pretreatment was performed in the presence of Ca2+, EP formation from ATP was only slightly retarded, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was ADP-sensitive. EP formation from Pi after chelating of Ca2+ was quite slow, whereas EP once formed was in rapid equilibrium with Pi of the medium. On the other hand, when the pretreatment was performed in the absence of Ca2+, EP formation from ATP was extremely slow, but EP once formed was in rapid dynamic equilibrium with ATP of the medium. EP formation from Pi was very fast, and this EP was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Ca2+-affinity form and the low Ca2+-affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This suggests that interactions between the enzyme protein and the surrounding lipids could play a crucial role in this isomerization.     

Mg(II) binding by bovine prothrombin fragment 1 via equilibrium dialysis and the relative roles of Mg(II) and Ca(II) in blood coagulation

The first direct equilibrium dialysis titration of the blood coagulation protein bovine prothrombin fragment 1 with Mg(II) is presented. Fragment 1 has fewer thermodynamic binding sites for Mg(II) than Ca(II), less overall binding affinity, and significantly less cooperativity. Several nonlinear curve fitting models were tested for describing the binding of fragment 1 with Mg(II), Ca(II), and mixed metal binding data. The Mg(II) data is represented by essentially five equivalent, noninteracting sites; for Ca(II), a model with three tight, cooperative sites and four "loose", equal affinity, noninteracting sites provides the best model. Based on the reported equilibrium dialysis data and in conjunction with other experimental data, a model for the binding of Ca(II) and Mg(II) to bovine prothrombin fragment 1 is proposed. The key difference between the binding of these divalent ions is that Ca(II) apparently causes a specific conformational change reflected by the cooperativity observed in the Ca(II) titration. The binding of Ca(II) ions to the three tight, cooperative sites establishes a conformation that is essential for phospholipid X Ca(II) X protein binding. The filling of the loose sites with Ca(II) ions leads to charge reduction and subsequent phospholipid X Ca(II) X protein complex interaction. Binding of Mg(II) to bovine prothrombin fragment 1 does not yield a complex with the necessary phospholipid-binding conformation. However, Mg(II) is apparently capable of stabilizing the Ca(II) conformation as is observed in the mixed metal ion binding data and the synergism in thrombin formation.     

Divalent cation binding to the high- and low-affinity sites on G-actin

Metal binding to skeletal muscle G-actin has been assessed by equilibrium dialysis using 45Ca2+ and by kinetic measurements of the increase in the fluorescence of N-acetyl-N'-(5-sulfo-1-naphthyl)-ethylenediamine-labeled actin. Two classes of cation binding sites were found on G-actin which could be separated on the basis of their Ca2+ affinity: a single high-affinity site with a Kd considerably less than 1 microM and three identical moderate-affinity binding sites with a Kd of 18 microM. The data for the Mg2+-induced fluorescence enhancement of actin labeled with N-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine support a previously suggested mechanism [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886] in which Ca2+ is replaced by Mg2+ at the moderate affinity site(s), followed by a slow actin isomerization. This isomerization occurs independently of Ca2+ release from the high-affinity site. The fluorescence data do not support a mechanism in which this isomerization is directly related to Ca2+ release from the high-affinity site. Fluorescence changes of labeled actin associated with adding metal chelators are complex and do not reflect the same change induced by Mg2+ addition. Fluorescence changes in the labeled actin have also been observed for the addition of Cd2+ or Mn2+ instead of Mg2+. It is proposed actin may undergo a host of subtle conformational changes dependent on the divalent cation bound. We have also developed a method by which progress curves of a given reaction can be analyzed by nonlinear regression fitting of kinetic simulations to experimental reaction time courses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ dependence of the interactions between protein C, thrombin, and the elastase fragment of thrombomodulin. Analysis by ultracentrifugation.

The two-way and three-way interactions among active-site-blocked bovine thrombin, bovine protein C, and the elastase fragment of rabbit thrombomodulin (elTM) were examined by analytical ultracentrifugation at 23.3 degrees C in 100 mM NaCl, 50 mM Tris (pH 7.65), and 1 mM benzamidine, in the presence of 0 to 5 mM calcium chloride. Thrombin and elTM form a tight (Kd less than 10(-8) M) 1:1 complex in the absence of Ca2+ that weakens with the addition of Ca2+ (Kd approximately 4 microM in 5 mM Ca2+). Without Ca2+, thrombin and protein C form a 1:1 complex (Kd approximately 1 microM) and what appears to be a 1:2 thrombin-protein C complex. The Kd for the 1:1 complex weakens over 100-fold in 5 mM CaCl2. Protein C and elTM form a Ca(2+)-independent 1:1 complex (Kd approximately 80 microM). Nearly identical binding to thrombin and elTM is observed when active-site-blocked activated bovine protein C is substituted for protein C. Thrombin inhibited by diisopropyl fluorophosphate and thrombin inhibited by a tripeptide chloromethyl ketone exhibited identical behavior in binding experiments, suggesting that the accessibility of protein C to the substrate recognition cleft of these two forms of thrombin is nearly equal. Human protein C binds with lower affinity than bovine protein C. Ternary mixtures also were examined. Protein C, elTM, and thrombin form a 1:1:1 complex which dissociates with increasing [Ca2+]. In the absence of Ca2+, protein C binds to the elTM-thrombin complex with an apparent Kd approximately 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of Ca2+-binding constants of proteins and presentation of the CaLigator software

The complexity of Ca2+ cell signaling is dependent on a plethoria of Ca2+-binding proteins that respond to signals in different ranges of Ca2+ concentrations. Since the function of these proteins is directly coupled to their Ca2+-binding properties, there is a need for accurately determined equilibrium Ca2+-binding constants. In this work we outline the experimental techniques available to determine Ca2+-binding constants in proteins, derive the models used to describe the binding, and present CaLigator, software for least-square fitting directly to the measured quantity. The use of the software is illustrated for Ca2+-binding data obtained for two deamidated forms of calbindin D(9k), either an isospartate-56 (beta form) or a normal Asp-56 (alpha form). Here, the Ca2+-binding properties of the two isoforms have been studied using the chelator method. The alpha form shows similar Ca2+-binding properties to the wild type while the beta form has lost both cooperativety and affinity.     

The role of calcium ions in factor X activation by thrombin E192Q.

Despite considerable sequence similarities, blood coagulation serine proteases exhibit remarkable specificity with respect to which zymogen they activate. The basis for this specificity presumably involves recognition of a short sequence within the extended binding pocket of the enzyme, other interactions remote from the catalytic groove, and modulation by a definite protein cofactor. In addition, Ca2+ plays a major role in most activation processes, but, because both the enzyme and its substrate interact with Ca2+, whether Ca2+ influences the substrate, the enzyme, or both remains an open question. Thrombin is not a factor X-activating enzyme, but when Glu192, 3 residues remote from the active Ser195, is replaced with glutamine, the resultant serine protease (E192Q) becomes a bovine, but not human, factor X activator. Kinetic experiments with peptides corresponding to human and bovine factor X activating sites reveal that threonine at position P2 in human (versus a valine in bovine) accounts for the species specificity. Substitution of the threonine in P2 of the human sequence with valine allows E192Q to cleave the human peptide whereas substitution of the valine in P2 of the bovine sequence with threonine hinders E192Q catalysis. Thrombin has no high affinity Ca2+ binding sites, and E192Q proteolysis of these peptides is not altered by Ca2+. The influence of Ca2+ in E192Q-mediated factor X activation provides therefore new insights into the role of the different Ca2+ binding sites in factor X. With factor X as substrate, the addition of Ca2+ enhances Kcat 4-fold but increases Km 10-fold. When the vitamin K-dependent gamma-carboxyglutamic acid domain of factor X is removed, the Km remains high with or without Ca2+ whereas Kcat still increases upon addition of the metal ion. These results suggest that factor X undergoes two metal-dependent suggest that factor X undergoes two metal-dependent transitions that influence the presentation of the activation site to activators.     

Influence of association and of positive inotropic drugs on calcium binding to cardiac troponin C

Isolated bovine cardiac troponin C forms dimers in presence of Mg2+ dependent on the protein concentration which has been determined by sedimentation velocity and sedimentation equilibrium. Dimer formation is correlated with a decrease in Ca2+ affinity. Positive inotropic drugs (benzimidazol derivatives) influence Ca2+ sensitivity either by changing the association state or by affecting the Ca2+ binding properties directly.     

Effects of pH, temperature and Ca2+ content on the conformation of alpha-lactalbumin in a medium modelling physiological conditions

Data obtained by the intrinsic protein fluorescence technique showed that, in addition to Ca2+ and Mg2+ ions, bovine alpha-lactalbumin also binds physiologically significant Na+ and K+ ions, the nucleotides ATP, ADP, UTP, UDP and UDP-galactose. The release of the bound Ca2+ ions from the protein in a medium modelling physiological conditions (containing Mg2+, Na+, K+, ATP and ADP in physiological concentrations) induced a transition of the protein from the native state of the Ca2+-loaded form to a state which is a mixture of native and and thermally changed states of the apo- and metal bound forms. Any variations in temperature result in changes in the populations of these states. This may be associated with some Ca2+ and temperature dependent regulation of the protein function. Variations of pH within the physiological limits had little influence on the conformation of both Ca2+-loaded and Ca2+-free alpha-lactalbumin.     

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.     

Fluorescence studies of the calcium binding to whiting (Gadus merlangus) parvalbumin

The calcium binding by parvalbumin of whiting (Gadus merlangus) has been studied using tryptophanyl fluorescence characteristics. Titration of Ca2+-free parvalbumin with Ca2+ leads to a very pronounced blue shift, narrowing and intensification of the fluorescence spectrum. These spectral changs proceed in two stages reflecting the existence of at least three forms which can be interpreted as (a) the protein without Ca2+, (b) with one Ca2+ and (c) with two bound Ca2+ ions/molecule. The fluorescence of these forms has been identified and the fluorescence spectra measured at varied Ca2+ concentrations were resolved into three components corresponding to these spectral forms. The dependence of the relative concentration of the three fomrs on Ca2+ concentrations agree well with the two-step binding of Ca2+ to parvalbumin: Protein + Ca in equilibrium K1 protein x Ca; Protein x Ca + Ca in equilibrium K2 Ca x protein x Ca. The equilibrium binding constants K1 and K2 obtained by the computer fit are approximately 5 X 10(8) M-1 and 6 X 10(6) M-1. This scheme and the K1 and K2 value are in a good agreement with the independent experimental data resulting from EGTA titration of Ca2+-saturated parvalbumin and pH titratin of parvalbumin in the presence of EGTA and CA2+.     

Equilibrium and kinetics of the folding and unfolding of canine milk lysozyme

The equilibrium and kinetics of canine milk lysozyme folding/unfolding were studied by peptide and aromatic circular dichroism and tryptophan fluorescence spectroscopy. The Ca2+-free apo form of the protein exhibited a three-state equilibrium unfolding, in which the molten globule state is well populated as an unfolding intermediate. A rigorous analysis of holo protein unfolding, including the data from the kinetic refolding experiments, revealed that the holo protein also underwent three-state unfolding with the same molten globule intermediate. Although the observed kinetic refolding curves of both forms were single-exponential, a burst-phase change in the peptide ellipticity was observed in both forms, and the burst-phase intermediates of both forms were identical to each other with respect to their stability, indicating that the intermediate does not bind Ca2+. This intermediate was also shown to be identical to the molten globule state observed at equilibrium. The phi-value analysis, based on the effect of Ca2+ on the folding and unfolding rate constants, showed that the Ca2+-binding site was not yet organized in the transition state of folding. A comparison of the result with that previously reported for alpha-lactalbumin indicated that the folding initiation site is different between canine milk lysozyme and alpha-lactalbumin, and hence, the folding pathways must be different between the two proteins. These results thus provide an example of the phenomenon wherein proteins that are very homologous to each other take different folding pathways. It is also shown that the native state of the apo form is composed of at least two species that interconvert.     

Interaction of Trimeresurus flavoviridis phospholipase A2 and its fragment with calcium ion

Dimeric T. flavoviridis phospholipase A2 has been studied in terms of the interaction with essential Ca2+ by equilibrium gel filtration, ultraviolet difference spectroscopy, fluorescence measurements, and chemical modifications with p-bromophenacyl bromide. The subunit bound to Ca2+ with a 1:1 molar ratio and no cooperative binding was observed. The hypochromic effect produced upon the binding of Ca2+ is due to perturbation of (a) specific tryptophan residue(s) located in the vicinity of the active site and appears to be characteristic of this enzyme. On the basis of the pH dependence of the dissociation constants, it has been found that the alpha-amino group (pKa 8.7) controls the binding of Ca2+. Deprotonation of the alpha-amino group is possibly accompanied by conformational transition to the active form which is able to bind Ca2+. This is in contrast to the case of bovine pancreatic phospholipase A2 in which Asp-49 (pKa 5.2) is responsible for the metal ion binding (Fleer et al. (1981) Eur. J. Biochem. 113, 283-288). Des-octapeptide(1-8)-phospholipase A2 (L-fragment) was found to be capable of binding Ca2+ under the control of a group with a pKa of 7.6. This pKa value was similar to an apparent pKa of 7.5 determined for the histidine residue in the active site of the native enzyme by way of p-bromophenacyl bromide modification. It appears that the N-terminal (octapeptide) sequence affects the binding mode of Ca2+, possibly because of conformational transition arising from its removal. The reinvestigation showed that the N-terminal octapeptide sequence is Gly-Leu-Trp-Gln-Phe-Glu-Asn-Met.     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

Kinetics of sulfate transport by Penicillium notatum. Interactions of sulfate, protons, and calcium.

The active transport of inorganic sulfate by an ATP sulfurylase-negative strain of Penicillium notatum is promoted by H+ ions and metal ions (divalent metal ions being more effective than monovalent metal ions). Initial velocity studies suggest that H+ and SO4(2-) add to the carrier in an ordered sequence (H+ before SO4(2-)), with H+ at equilibrium with free carrier and carrier-H+ complex. The linear reciprocal plots and replots suggest a 1:1 stoichiometry between H+ and SO4(2-). Ca2+ and other divalent metal ions stimulate sulfate transport markedly in buffered suspensions of low ionic strength. The kinetics of the Ca2+/SO4(2-) interaction suggest that Ca2+ (like H+) adds to the carrier before SO4(2-) and is at equilibrium with free carrier and carrier-Ca2+ complex. The linear reciprocal plots and replots indicate a 1:1 stoichiometry between Ca2+ and SO4(2-). Thus the fully loaded carrier-SO4(2-) -Ca2+ -H+ complex has a net positive charge relative to that of the free carrier, a fact consistent with the chemiosmotic hypothesis of membrane transport. The kinetics of the H+/Ca2+ interaction point to a random A-B (rapid equilibrium), ordered C sequence with A = H+, B = Ca2+, and C = SO4(2-). Selenate (an alternate substrate competitive with sulfate) is an uncompetitive inhibitor with respect to Ca2+, in agreement with the suggested mechanism. Internal charge balance is not accomplished by a stoichiometric coaccumulation of Ca2+ and SO4(2-). Sulfate transport does, however, promote 45Ca2+ uptake. A significant fraction of the added Ca2+ is bound by the mycelial surface. Binding is extremely rapid, but reversible.