Author index for volume 93

The presence of Ca²⁺ is essential for survival in culture of fully grown oocytes isolated from mouse ovaries but not for survival of small, meiotically incompetent oocytes, metaphase II oocytes, and early embryos. Ninety percent of fully grown ovarian oocytes die within 2 hr when cultured in calcium-free medium (CFM). CFM death does not occur if other cations (1 mM La³⁺ or 10 mM Sr²⁺, but not 12 mM Mg²⁺ nor 1 mM D-600) replace Ca²⁺ in the medium. Sensitivity to CFM is progressively acquired by the oocyte during the growth phase, in parallel with the acquisition of meiotic competence, and is lost after 2 hr of culture in the presence of at least 0.5 mM Ca²⁺. The loss of sensitivity to CFM during in vitro culture is not related to the concomitant spontaneous resumption of meiosis, since the oocyte becomes resistant to CFM even if germinal vesicle breakdown is prevented by the addition of dibutyryl cAMP to the culture medium. Some hypotheses are put forward to explain the peculiar and transient high calcium requirements of fully grown oocytes.     

Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae

The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na⁺ and K⁺ was demonstrated, with maximum stimulation at 20–40 mM concentrations. The divalent cations Mg²⁺ and Ca²⁺ stimulated proteolytic activity at low concentrations (between 0 and 10 mM) but inhibited activity at higher concentrations. The divalent cations Zn²⁺, Cu²⁺, Fe²⁺, and Co²⁺ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.     

Effect of an anti-tumor platinum complex,Pt(II)diaminotoluene,on mitochondrial membrane properties

The effects of platinum complexes, selected for their potent anti-tumor activities, have been studied on rat liver mitochondria. Among the mitochondrial properties which have been studied, the most marked effects of platinum complexes were obtained on functions linked to the inner membrane.cis-Pt(II)(3,4-diaminotoluene) dichloride is shown to stimulate state 4 respiration. It inhibits the phosphate transport into mitochondria, decreases the accumulation of Ca²⁺, and induces a more rapid release of the accumulated Ca²⁺. A release of Mg²⁺ from mitochondria incubated in the absence of added divalent cations, and an efflux of divalent cations from mitochondrial membranes are also observed.All these results indicate a profound modification of the permeability of mitochondrial membrane.     

N-type calcium channels are involved in the dopamine releasing effect of nicotine

Mouse striatum was incubated with [³H]dopamine ([³H]DA) and superfused with and the tritium efflux induced by nicotine, electrical stimulation, or simultaneous nicotine and electrical stimulation was measured, to characterize the role of different Ca²⁺ channels in the transmitter release. Nicotine stimulation and electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotine or electrical stimulation increases the release of [³H]DA and that the outflow of³H-labeled metabolites was similar with the two different stimulation procedures. Removal of Ca²⁺ from the superfusate resulted in a marked reduction in the tritium release evoked by nicotine, whereas the electrical stimulation-evoked tritium release was completely dependent on external Ca²⁺. The L-and N-type calcium channel blockers omega-conotoxin GVIA and Cd²⁺ inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the L-type and T-type channel blockers diltiazem and Ni²⁺ did not alter release of [³H]DA. We conclude that N-type voltage-sensitive Ca²⁺ channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as Ca²⁺ and N-type voltage-sensitive calcium channels may simultaneously open up, and they additively increase free intracellular Ca²⁺ concentration.     

Divalent cation influx and calcium homeostasis in germinal vesicle mouse oocytes

Prior to maturation, mouse oocytes are arrested at the germinal vesicle (GV) stage during which they experience constitutive calcium (Ca²⁺) influx and spontaneous Ca²⁺ oscillations. The oscillations cease during maturation but Ca²⁺ influx continues, as the oocytes’ internal stores attain maximal content at the culmination of maturation, the metaphase II stage. The identity of the channel(s) that underlie this Ca²⁺ influx has not been completely determined. GV and matured oocytes are known to express three Ca²⁺ channels, Ca_V3.2, TRPV3 and TRPM7, but females null for each of these channels are fertile and their oocytes display minor modifications in Ca²⁺ homeostasis, suggesting a complex regulation of Ca²⁺ influx. To define the contribution of these channels at the GV stage, we used different divalent cations, pharmacological inhibitors and genetic models. We found that the three channels are active at this stage. Ca_V3.2 and TRPM7 channels contributed the majority of Ca²⁺ influx, as inhibitors and oocytes from homologous knockout (KO) lines showed severely reduced Ca²⁺ entry. Sr²⁺ influx was promoted by Ca_V3.2 channels, as Sr²⁺ oscillations were negligible in Ca_V3.2-KO oocytes but robust in control and Trpv3-KO GV oocytes. Mn²⁺ entry relied on expression of Ca_V3.2 and TRPM7 channels, but Ni²⁺ entry depended on the latter. Ca_V3.2 and TRPV3 channels combined to fill the Ca²⁺ stores, although Ca_V3.2 was the most impactful. Studies with pharmacological inhibitors effectively blocked the influx of divalent cations, but displayed off-target effects, and occasionally agonist-like properties. In conclusion, GV oocytes express channels mediating Ca²⁺ and other divalent cation influx that are pivotal for fertilization and early development. These channels may serve as targets for intervention to improve the success of assisted reproductive technologies.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Hormonal control of meiosis : In vitro induced release of calcium ions from the plasma membrane in starfish oocytes

Using the photoprotein aequorin as a marker, we found that the Ca²⁺ release, which we demonstrated to occur from the inner part of the plasma membrane after hormonal stimulation of intact starfish oocytes can also be recorded in vitro. Our acellular system was prepared from cortices of Marthasterias glacialis oocytes isolated in Ca²⁺-free medium and extracted with Triton X-100. At 0–4 °C, the 100 000 g pellet obtained from this plasma membrane-rich extract responds in less than 0.1 sec to the hormone 1-methyladenine, its biological active analogues and mimetics, whereas supernatant exhibits a quite lower activity which is no longer present in the head fraction recovered after dialysis through Diaflo UM 2 filters. The in vitro calcium response is competitively inhibited by various agents which, in the same concentration range, act on meiosis reinitiation and free calcium release by the living oocyte. These and previous data are further discussed taking into account the overall chain of events which leads to the release of meiosis inhibition.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Stimulation of the insulin secretory mechanism following barium accumulation in pancreatic β-cells

Electrothermal atomic absorption spectroscopy was employed for measuring barium in β-cell-rich pancreatic islets microdissected from ob/ob-mice. Both the uptake and efflux of barium displayed two distinct phases. There was a 4-fold accumulation of barium into intracellular stores when its extracellular concentration was 0.26 mM. Unlike divalent cations with more extensive intracellular accumulation, the washout of Ba²⁺ was not inhibited by d-glucose. Ba²⁺ served as a substitute for Ca²⁺ both in maintaining the glucose metabolism after removal of extracellular Ca²⁺ and making it possible for glucose to stimulate insulin release. Furthermore, Ba²⁺ elicited insulin release in the absence of glucose and other secretagogues. The latter effect was reversible and was markedly potentiated under conditions known to increase the β-cell content of cyclic AMP. It is likely that the observed actions of Ba²⁺ are mediated by Ca²⁺, since Ca²⁺-dependent regulatory proteins, such as calmodulin, apparently cannot bind Ba²⁺ specifically.     

The influence of monovalent cations upon influx and efflux of Ca2+ in barley roots

The effects of monovalent cations, inhibitors of metabolism dinitrophenol (DNP), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and KCN and temperature variations upon Ca²⁺ fluxes in intact roots of barley (Hordeum vulgare L. cv. Fergus and Herta) seedlings were investigated. ⁴⁵Ca²⁺ influx was depressed in CaSO₄-grown (low-salt) plants by the presence of NH₄⁺, K⁺, or Na⁺ in the uptake medium. In contrast Ca²⁺ influx was slightly increased by Li⁺. In low-salt roots pretreated with KCN and in roots preloaded with K⁺ (high-K⁺ plants), the presence of K⁺ in the medium had no significant effect on Ca²⁺ influx, while in roots preloaded with Na⁺, the presence of K⁺ in the medium depressed Ca²⁺ influx. In absolute terms, Ca²⁺ influx was significantly greater in high-salt (both K⁺ or Na⁺ preloaded) than in low-salt roots.Patterns of ⁴⁵Ca²⁺ efflux in the absence and in the presence of K⁺, NH₄⁺, or Li⁺ in the external medium showed that these monovalent cations caused stimulation of ⁴⁵Ca²⁺ efflux both from the cytoplasmic and vacuolar phases.It was noted that these modifications of Ca²⁺ fluxes by monovalent cations are transient and characteristic of a transitional stage of cation uptake by low-salt roots. We conclude that, together with stimulated active H⁺ efflux (another characteristic of this transitional stage), modifications of Ca²⁺ fluxes during monovalent cation uptake by low-salt roots is a response directed towards the maintenance of electrical neutrality.Determination of net fluxes revealed that the plants were close to Ca²⁺ flux equilibrium in the growth medium (0.5 mM CaSO₄). Transfer of these plants to 0.5 mM CaSO₄ + 0.25 mM K₂SO₄ caused a net release of CA²⁺ into the external medium.     

Induction of amphibian oocyte maturation by polyvalent cations and alkaline pH in the absence of potassium ions.

The maturation of Xenopus laevis oocytes was studied in media free of added potassium salts. Under these conditions maturation could be triggered by 1 mM Mn²⁺ and La³⁺ and, to a lesser extent, by 2–4 mM Ca²⁺ and Mg²⁺. Maturation induced by 1.5 mM Mn²⁺ was inhibited by K⁺ concentrations above 0.25 mM. Potassium was inhibitory when added up to 2 hr before germinal vesicle breakdown occurred. In potassium-free media, maturation could be induced by incubation of oocytes under mild alkaline media (pH 8.5–9). A high percentage of medium-sized oocytes (stage IV according to Dumont) was induced to mature by progesterone in the absence of potassium. Maturation of oocytes in potassium-free media was normal by the criteria of germinal vesicle breakdown, production of maturation promoting factor, vitelline membrane activation, and inhibition by known maturation inhibitors.     

Alkaline pH and Internal Calcium Increase Na+ and K+ Effluxes in LK Sheep Red Blood Cells in Cl−-free Solutions

We examined the effects of pH, internal ionized Ca (Ca²⁺ _i ), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K⁺ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K⁺ phenotypes. In LK SRBCs the K⁺ efflux was higher at pH 9.0 (350%) than at pHs 7.4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K⁺ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K⁺ efflux was activated (100%) when Ca²⁺ _i was increased (+A23187, +Ca²⁺ _o ) into the μm range. However, in comparison to human red blood cells, the Ca²⁺ _i -induced increase in K⁺ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na⁺ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca²⁺ _i . In contrast, in HK SRBCs the K⁺ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca²⁺ _i . These studies suggest the presence in LK SRBCs, of at least two pathways for Cl⁻-independent K⁺ and Na⁺ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca²⁺ _i . Received: 23 May 1996/Revised: 6 December 1996     

Divalent cations and inorganic phosphate metabolism in starved Tetrahymena

Tetrahymena pyriformis maintained under starvation conditions release orthophosphate into the suspension medium. Ca²⁺ and/or Mg²⁺ addition reduced the amount of orthophosphate excreted during a 3-h period. Cellular orthophosphate levels were not altered by divalent cation supplements; however, an increase in the pyrophosphate content was observed which was equivalent in amount to the reduction in orthophosphate efflux. These observations suggest that divalent cations are important not only in the acquisition of phosphorus during growth but also in the conservation of this element during starvation.     

Use-dependent block with tetrodotoxin and saxitoxin at frog Ranvier nodes II. Extrinsic influence of cations

Use-dependent declines of Na⁺ currents in myelinated frog nerve fibres were measured during a train of depolarizing pulses in solutions containing tetrodotoxin (TTX) or saxitoxin (STX). The following effects of external monovalent (Na⁺), divalent (Ca²⁺, Mg²⁺) and trivalent (La²⁺) cations on use dependence were found: Increasing the Ca²⁺ concentration from 2 to 8 mM shifts its voltage dependence by 20 mV whereas no significant use-dependent decline occurred at 0.2 mM Ca²⁺. Doubling the external Na⁺ concentration in 0.2 mM Ca²⁺ solutions did not initiate phasic block. External Mg²⁺ ions induced a smaller, and La²⁺ ions a larger, use dependence. The time constants of the current decline were 4-fold greater in 1.08 mM La²⁺. The static block of Na⁺ currents by La³⁺ could be directly demonstrated by the relief of block during a train of pulses. The results are qualitatively explained by a toxin binding site at the Na⁺ channel whose affinity for TTX or STX depends oni) the gating conformation of the channel, probably the inactivation andii) the occupancy of a blocking site by di- or trivalent external cations.     

Membrane fusion of secretory vesicles and liposomes Two different types of fusion

Secretory vesicles isolated from adrenal medulla were found to fuse in vitro in response to incubation with Ca²⁺. Intervesicular fusion was detected by electron microscopy and was indicated by the appearance of twinned vesicles in freeze-fractured suspensions of vesicles and in thin-sectioned pellet. Two types of fusion could be distinguished: Type I, occurring between 10^?7 M and 10^?4 M Ca²⁺, was specific for Ca²⁺, was inhibited by other divalent cations and was abolished by pretreatment of vesicles with glutaraldehyde, neuraminidase or trypsin. Fusion type I was linear with temperature. A second type of intervesicular fusion was elicited by Ca²⁺ in concentrations higher than 2.5 mM and was morphologically characterized by multiple fusions of secretory vesicles. This type of fusion was found to be similar to fusion of liposomes prepared from the membrane lipids of adrenal medullary secretory vesicles: Ca²⁺ could be replaced by other divalent cations, the effect of different divalent cations was additive and pretreatments attacking membrane proteins were ineffective. Fusion type II of intact secretory vesicles as well as liposome fusion was discontinuous with temperature. Liposome fusion could be detected within 35 ms and persisted for 180 min. Using liposomes containing defined Ca²⁺ concentrations we have not found a major influence of Ca²⁺ asymmetry on fusion. Incorporation of the ganglioside GM₃, which is present in the membranes of intact adrenal medullary secretory vesicles did not change the properties of liposomes fusion. Using a Ca²⁺-selective electrode we have identified in secretory vesicle membranes both high affinity binding sites for Ca²⁺ ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6 · 10}{}^{\mathrm{?6}}\text{M}$) and low affinity sites ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.2 · 10}{}^{\mathrm{?4}}\text{M}$).     

Relationships between hormone-induced calcium release and 86rubidium uptake stimulation in starfish oocytes

86Rubidium+ uptake, but not 86Rubidium efflux, is strongly stimulated after addition of the meiosis inducing hormone 1-methyladenine (1-MeAde) to prophase blocked oocytes of the starfish Marthasterias glacialis. This stimulation is a transient process which does not require the continuous presence of 1-MeAde and is elicited within 1 minute of contact. 1-MeAde and its biologically active structural analogs fully stimulate Rb+ uptake at concentrations which are about two orders of magnitude lower than those required to trigger meiosis reinitiation but which already release underthreshold levels of Ca2+ from the inner part of the plasma membrane. External Ca2+ concentrations effective in triggering meiosis reinitiation also stimulate Rb+ influx, while drugs like D600, theophyllin and caffein which suppress the hormone induced Ca2+ release, simultaneously preclude the stimulation of Rb+ uptake. Dithiothreitol (DTT) which mimicks 1-MeAde action in releasing Ca2+ and inducing meiosis acts both on the efflux and on active and passive Rb+ influxes. Ouabain, the classical inhibitor of the Na+, K+ pump does not preclude meiosis reinitiation under the influence of 1-MeAde, its agonists of mimetics. It suppresses the active component of Rb+ uptake both in control or stimulate oocytes. When applied only in preincubation before starting the hormone treatment, it cannot however inhibit the stimulation of Rb+ uptake, while basal pump inhibition is preserved. These results demonstrate that stimulation of the active Rb+ or K+ transport is not indispensable to meiosis reinitiation. They suggest moreover that the hormone induced Ca2+ release from the plasma membrane may be responsible for unmasking new ouabain sensitive transport sites.     

Ryanodine as a functional probe of the skeletal muscle sarcoplasmic reticulum Ca2+ release channel

Ryanodine is a neutral plant alkaloid which functions as a probe for an intracellular Ca²⁺ release channel (ryanodine receptor) in excitable tissues. Using [³H]ryanodine, a 30 S protein complex comprised of four polypeptides of M_r 565,000 has been isolated and functionally reconstituted into planar lipid bilayers. The effects of salt concentration and divalent cations on skeletal muscle sarcoplasmic reticulum [³H]ryanodine binding and Ca²⁺ release channel activity have been compared. These studies suggest that ryanodine is a good probe for investigating the function of the release channel.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH,cations and phosphate

The uptake of Ca²⁺ and Sr²⁺ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics.The rate of Ca²⁺ and Sr²⁺ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentrations of Ca²⁺ and Sr²⁺ at low pH may be explained by a decrease of the surface potential.The inhibition of Ca²⁺ and Sr²⁺ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane.Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

The ionic components of the current pulses generated by developing fucoid eggs.

Using a newly developed, extracellular vibrating electrode, we studied the ionic composition of the current pulses which traverse the developing Pelvetia embryo. External Na⁺, Mg²⁺, or SO₄^2?, are not needed for the first 20 min of pulsing. In fact, lowering external Na⁺ or Mg²⁺ (or K⁺) actually stimulates pulsing. Since tracer studies show that Ca²⁺ entry is speeded by Na⁺, Mg²⁺, or K⁺ reduction, these findings suggest that Ca²⁺ entry triggers pulsing. A sevenfold reduction in external Cl^? raises pulse amplitudes by 60%. Moreover, Cl^? is the only major ion with an equilibrium potential near the pulse reversal potential. These facts suggest that Cl^? efflux carries much of the “inward” current. We propose a model for pulsing in which increased Ca²⁺ within the growing tip opens Cl^? channels. The resulting Cl^? efflux slightly depolarizes the membrane and thus drives a balancing amount of K⁺ out. Thus, the pulses release KCl and serve to relieve excess turgor pressure. By letting Ca²⁺ into the growing tip, they should also strengthen the transcytoplasmic electrical field which is postulated to pull growth components toward this tip.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.