Reduced Ca(2+)-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum at low pH.

The purpose of this investigation was to determine the effects of reduced pH on Ca(2+)-induced Ca2+ release (CICR) from skeletal muscle sarcoplasmic reticulum (SR). Frog semitendinosus fiber bundles (1-3/bundle) were chemically skinned via saponin treatment (50 micrograms/mL, 20 min), which removes the sarcolemma and leaves the SR functional. The SR was first depleted of Ca2+ then loaded for 2 min at pCa (log free Ca2+ concentration) 6.6. CICR was then evoked by exposing the fibers to pCa 5-7 for 5-60 s. CICR was evoked both in the absence of ATP and Mg2+ and in the presence of beta, gamma-methyleneadenosine-5'-triphosphate (AMPPCP, a nonhydrolyzable form of ATP) and Mg2+. Ca2+ remaining in the SR was then assayed via caffeine (25 mM) contracture. In all cases, CICR evoked at pH 6.5 resulted in larger caffeine contractures than that evoked at 7.0, suggesting that more Ca2+ was released during CICR at the higher pH. Accordingly, rate constants for CICR were significantly greater at pH 7.0 than at pH 6.5. These results indicate that reduced pH depresses CICR from skeletal muscle SR.     

Ca2+ influx activated by low pH in Chlamydomonas

Cytosolic acidification stimulates an influx of Ca2+ which results in shedding of the two flagella of Chlamydomonas. Ca2+ influxes are also involved in the photoresponses of this alga, but it is not understood how the acidification-activated Ca2+ influx is distinguished from the Ca2+ influxes which mediate phototaxis and the photophobic response. The present study focuses on the deflagellation-inducing Ca2+ influx pathway. Influx occurs through an ion channel or transporter with low abundance or low permeability to Ca2+ (approximately 500 fmol/s/10(6) cells in 50 microM Ca2+). Ca2+ influx was potently blocked by Cd3+ (EC50 approximately 5 microM), but was insensitive to Cd2+ (Quarmby, L.M., and H.C. Hartzell. 1994. J. Cell Biol. 124:807) and organic blockers of Ca2+ channels including SKF-96365 (up to 100 microM) and flufenamic acid (up to 1 mM). Experiments with a flagella-less mutant (bald-2), isolated flagella, and a blocker of flagellar assembly (colchicine) indicated that the acidification-stimulated Ca2+ influx pathway is not localized to the flagellar membrane. The acid-stimulated influx pathway was transiently inactivated after cells shed their flagella. Inactivation did not occur in the deflagellation mutant, fa- 1, although acidification-stimulated Ca2+ influx was normal. This suggests that inactivation of this pathway in wild-type cells is probably not a direct consequence of acidification nor of Ca2+ influx, but may be related to deflagellation. Recovery of deflagellation- inducing Ca2+ influx occurred within 30 min after a 30 s exposure to acid and did not require flagellar assembly. The regulation, drug sensitivity, and subcellular localization identify acidification- stimulated Ca2+ influx as a specific Ca2+ entry pathway distinct from established Ca2+ channels.     

The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca²⁺ ions. Earlier studies suggested a critical role of one of the bound Ca²⁺ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca²⁺-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca²⁺-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca²⁺, perturbs a H-bonding network connecting this Ca²⁺ to the heme crevice that involves the distal water. The measured effects of Ca²⁺ depletion can be interpreted as supporting a structural role for the distal Ca²⁺ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.     

Formation of two types of low-spin heme in horseradish peroxidase isoenzyme A2 at low temperature

Electronic absorption, resonance Raman and EPR spectra are reported for ferric horseradish peroxidase isoenzyme A2 at neutral and alkaline pH together with its imidazole complex at 12 K. The data are compared with those obtained at room temperature. At neutral pH, lowering the temperature induces conformational changes with the formation of two types of low-spin hemes, a bis-histidyl type and a hydroxo type. The transition induced by lowering the temperature is accompanied by a change in the orientation of a vinyl substituent which appears less conjugated to the porphyrin macrocycle than at room temperature. At low temperature the low-spin hemes coexist with a quantum admixed spin species. All the forms are characterized by extremely high resonance Raman frequencies, indicating a contraction of the core size from that of the room temperature species. At alkaline pH, only one low-spin species is observed at both room and low temperatures, with a hydroxo ligand bound to the heme iron. The ν(Fe-OH) stretching mode has been assigned at 512 cm⁻¹, on the basis of the isotopic shift observed in D₂O and H₂ ¹⁸O. This relatively low frequency, together with the anomalous shift observed in deuterium, indicates that the hydrogen bonds between the oxygen atom and the distal residues are stronger than in metmyoglobin, but weaker than those of horseradish peroxidase isoenzyme C. This is in agreement with the lower tetragonality, determined from the EPR g values, of alkaline horseradish peroxidase isoenzyme A2 than of metmyoglobin. Received: 30 September 1999 / Accepted: 17 January 2000     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH

Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.     

Sensitized photooxidation of low spin horseradish peroxidase.

Horseradish peroxidase differs from most enzymes in that it is almost completely resistant to photodynamic action due to the paramagnetic ferric ion in the prosthetic group, heme. Chelation of horseradish peroxidase at the sixth coordination position of the iron with a cyanide or hydroxyl group converts it to a low spin diamagnetic state. Upon illumination with visible light with eosin Y, flavin mononucleotide or methylene blue as sensitizer, the low spin enzyme lost both peroxidative and oxidative activities with the same quantum yields. Several amino acid residues, including one histidine and one tyrosine were destroyed in the low spin enzyme after 60 min of illumination with eosin Y as sensitizer.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 microM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca2+ and decreased approx. 1000-times when the Ca2+ concentration was reduced from 112 to 0.5 microM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (CaiZ) decreases in the order of Ca3Z greater than Ca4Z greater than Ca2Z greater than or equal to CaZ. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca2+-ATPase in the range of 10(-7)-10(-6) M Ca2+, even at a calmodulin concentration of 5 microM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 microM, corresponding to 50-80-times the cellular concentration of Ca2+-ATPase, estimated to be approx. 10 nmol/h membrane protein. We therefore conclude that most of the calmodulin is dissociated from the Ca2+-transport ATPase in erythrocytes at the prevailing Ca2+ concentration (probably 10(-7)-10(-8) M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca2+-ATPase requires that the Ca2+ concentration rises to 10(-6)-10(-5) M.     

Zero-field splitting of Fe3+ in horseradish peroxidase and of Fe4+ in horseradish peroxidase compound I from electron spin relaxation data.

From the temperature dependence of the Orbach relaxation rate of the paramagnetic center in horseradish peroxidase (HRP), we deduce an excited-state energy of 40.9 +/- 1.1 K. Similar studies on the broad EPR signal of HRP compound I indicate a much weaker Orbach relaxation process involving an excited state at 36.8 +/- 2.5 K. The strength of the Orbach process in HRP-I is weaker than one would normally estimate by 2-4 orders of magnitude. This fact lends support to the model of HRP-I involving a spin 1/2 free radical coupled to a spin 1 Fe4+ heme iron via a weak exchange interaction. Such a system should exhibit an Orbach relaxation process involving delta E, the excited state of the Fe4+ ion, but reduced in strength by (Jyy/delta E)2, where Jyy is related to the strength of the exchange interaction between the two spin systems.     

pH dependence of carbon monoxide binding to ferrous horseradish peroxidase

The kinetic parameters of the reaction of horseradish peroxidase with CO have been determined at pH values between 10 and 3. At pH 7.0 the CO binding equilibrium constant L was measured using submicromolar concentrations of horseradish peroxidase; the value obtained corresponds to the ratio of the association and dissociation kinetic constants as expected for a simple binding mechanism to a monomeric hemeprotein. The CO association rate constant is pH-independent below pH 7, whereas in going from pH 7 to pH 11 a 2-fold increase can be detected, as previously reported (Kertesz, D., Antonini, E., Brunori, M., Wyman, J., and Zito, R. (1965) Biochemistry 4, 2672-2676). On the other hand, CO dissociation displays a peculiar pH rate profile characterized by a progressive decrease from pH 10 to pH 5 and by a very marked increase as the pH is further lowered to pH congruent to 3. Furthermore, the rate of CO dissociation is markedly enhanced in peroxidase reconstituted with protoheme dimethyl ester, suggesting a role of the propionates in the regulation of this process.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.

The "shell" model for Ca2(+)-inactivation of Ca2+ channels is based on the accumulation of Ca2+ in a macroscopic shell beneath the plasma membrane. The shell is filled by Ca2+ entering through open channels, with the elevated Ca2+ concentration inactivating both open and closed channels at a rate determined by how fast the shell is filled. In cells with low channel density, the high concentration Ca2+ "shell" degenerates into a collection of nonoverlapping "domains" localized near open channels. These domains form rapidly when channels open and disappear rapidly when channels close. We use this idea to develop a "domain" model for Ca2(+)-inactivation of Ca2+ channels. In this model the kinetics of formation of an inactivated state resulting from Ca2+ binding to open channels determines the inactivation rate, a mechanism identical with that which explains single-channel recordings on rabbit-mesenteric artery Ca2+ channels (Huang Y., J. M. Quayle, J. F. Worley, N. B. Standen, and M. T. Nelson. 1989. Biophys. J. 56:1023-1028). We show that the model correctly predicts five important features of the whole-cell Ca2(+)-inactivation for mouse pancreatic beta-cells (Plants, T. D. 1988. J. Physiol. 404:731-747) and that Ca2(+)-inactivation has only minor effects on the bursting electrical activity of these cells.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Regulation of Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase at limiting [Ca2+

The factors regulating Ca2+ transport by isolated sarcoplasmic reticulum (SR) vesicles have been studied using the fluorescent indicator Fluo-3 to monitor extravesicular free [Ca2+]. ATP, in the presence of 5 mM oxalate, which clamps intravesicular [Ca2+] at approximately 10 microM, induced a rapid decline in Fluo-3 fluorescence to reach a limiting steady state level. This corresponds to a residual medium [Ca2+] of 100 to 200 nM, and has been defined as [Ca2+]lim, whilst thermodynamic considerations predict a level of less than 1 nM. This value is similar to that measured in intact muscle with Ca2+ fluophores, where it is presumed that sarcoplasmic free [Ca2+] is a balance between pump and leaks. Fluorescence of Fluo-3 at [Ca2+]lim was decreased 70% to 80% by histidine, imidazole and cysteine. The K0.5 value for histidine was 3 mM, suggesting that residual [Ca2+]lim fluorescence is due to Zn2+. The level of Zn2+ in preparations of SR vesicles, measured by atomic absorption, was 0.47+/-0.04 nmol/mg, corresponding to 0.1 mol per mol Ca-ATPase. This is in agreement with findings of Papp et al. (Arch. Biochem. Biophys., 243 (1985) 254-263). Histidine, 20 mM, included in the buffer, gave a corrected value for [Ca2+]lim of 49+/-1.8 nM, which is still higher than predicted on thermodynamic grounds. A possible 'pump/leak' mechanism was tested by the effects of varying active Ca2+ transport 1 to 2 orders with temperature and pH. [Ca2+]lim remained relatively constant under these conditions. Alternate substrates acetyl phosphate and p-NPP gave similar [Ca2+]lim levels even though the latter substrate supported transport 500-fold slower than with ATP. In fact, [Ca2+]lim was lower with 10 mM p-NPP than with 5 mM ATP. The magnitude of passive efflux from Ca-oxalate loaded SR during the steady state of [Ca2+]lim was estimated by the unidirectional flux of 45Ca2+, and directly, following depletion of ATP, by measuring release of 40Ca2+, and was 0.02% of Vmax. Constant infusion of CaCl2 at [Ca2+]lim resulted in a new steady state, in which active transport into SR vesicles balances the infusion rate. Varying infusion rates allows determination of [Ca2+]-dependence of transport in the absence of chelating agents. Parameters of non-linear regression were Vmax=853 nmol/min per mg, K0.5(Ca)=279 nM, and nH(Ca)=1.89. Since conditions employed in this study are similar to those in the sarcoplasm of relaxed muscle, it is suggested that histidine, added to media in studies of intracellular Ca2+ transients, and in the relaxed state, will minimise contribution of Zn2+ to fluophore fluorescence, since it occurs at levels predicted in this study to cause significant overestimation of cytoplasmic free [Ca2+] in the relaxed state. Similar precautions may apply to non-muscle cells as well. This study also suggests that [Ca2+]lim in the resting state is a characteristic feature of Ca2+ pump function, rather than a balance between active transport and passive leakage pathways.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity Ca2+-calmodulin targets

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin). However, little is known about the dynamics of how CaM targets process the Ca2+ signals to generate appropriate biological responses in the heart. We hypothesized that the different affinities of CaM targets for the Ca2+-bound CaM (Ca2+-CaM) shape their actions through dynamic and tonic interactions in response to the repetitive Ca2+ signals in myocytes. To test our hypothesis, we used two fluorescence resonance energy transfer-based biosensors, BsCaM-45 (Kd = approximately 45 nm) and BsCaM-2 (Kd = approximately 2 nm), to monitor the real time Ca2+-CaM dynamics at low and high affinity CaM targets in paced adult ventricular myocytes. Compared with BsCaM-2, BsCaM-45 tracks the beat-to-beat Ca2+-CaM alterations more closely following the Ca2+ oscillations at each myocyte contraction. When pacing frequency is raised from 0.1 to 1.0 Hz, the higher affinity BsCaM-2 demonstrates significant elevation of diastolic Ca2+-CaM binding compared with the lower affinity BsCaM-45. Biochemically detailed computational models of Ca2+-CaM biosensors in beating cardiac myocytes revealed that the different Ca2+-CaM binding affinities of BsCaM-2 and BsCaM-45 are sufficient to predict their differing kinetics and diastolic integration. Thus, data from both experiments and computational modeling suggest that CaM targets with low versus high Ca2+-CaM affinities (like CaM-dependent kinase versus calcineurin) respond differentially to the same Ca2+ signal (phasic versus integrating), presumably tuned appropriately for their respective and distinct Ca2+ signaling pathways.     

The pH induced modification of the electrophoretic mobilities of horseradish peroxidase isozymes

Isozymes of horseradish peroxidase may be generated from preexisting forms of the enzyme by incubation at 4 °C in solutions with pH's of 7 or higher. Isozymes generated in this manner express an apparent net increase in negative charge compared to the original form of the enzymes. This is evidenced by an increase in anodic electrophoretic mobility and a decrease in isoelectric point. The generation of new isozymes of peroxidase by such treatment alters the isozyme distribution pattern considerably, but there is no net change in total peroxidase activity present in the extract if pH's of 10.0 or lower are used. The generated peroxidase isozymes are formed irreversibly; neither retitration of extracts to a lower pH nor heat treatment will restore the original peroxidase isozymes.     

Demonstration of Na+-dependent Ca2+ efflux using low concentrations of fluorometric Ca2+ probes

The effects of different concentrations of the fluorometric Ca2+ probes, fura-2 and indo-1, on Ca2+ transients in cultured rat aortic smooth muscle cells were examined. When stimulated with the agonists, angiotensin II and arginine vasopressin, cells incubated with low concentrations of fura-2 or indo-1 (less than 1 microM) produced Ca2+ transients characterized by a small increase followed by a dramatic decrease in fluorescence below the original baseline. This effect of agonists was concentration-dependent, reversible, and blocked by receptor antagonists. In contrast to the agonists, stimulation of Ca2+ transients with depolarizing concentrations of K+ or with caffeine did not produce decreases in fluorescence and Ca2+ levels at any loading concentration of probe. The decrease in Ca2+ observed with agonists was dependent on the presence of extracellular Na+. These data suggest that under certain loading conditions, fluorescent Ca2+ indicators measure agonist-stimulated Ca2+ efflux mediated by a Na+/Ca2+ exchange mechanism.     

Mechanism-based irreversible inactivation of horseradish peroxidase at 500 MPa

The effects of high-pressure treatment on the reaction rates of horseradish peroxidase (HRP) with guaethol or guaiacol as a hydrogen donor were evaluated from direct transmission measurements in a high-pressure optical cell at 435 nm. Peroxidases are known to be very barostable and insensitive to heat. With guaethol the reaction velocity was independent of pressure up to 500 MPa, but with guaiacol the cytochrome c oxidase underwent a mechanism-based irreversible inhibition of catalytic activity when subjected to pressure; in the resting states (fully oxidized or reduced), it was insensitive to pressure. The enzyme inactivation took place with an inactivation rate constant of 5.15 x 10(-1) min(-1) at 500 MPa, 25 degrees C and pH 7. The degree of inactivation was correlated to the concentration of guaiacol. This is the first report on a mechanism-based pressure inactivation of HRP triggered at moderate pressure and temperature and mediated by the hydrogen donor.