Nonselective cation channel activated by patch excision from lobster olfactory receptor neurons

Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na⁺, K⁺ and Cs⁺, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na⁺ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg²⁺ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg²⁺ block of Na^– permeation, and demonstrating the channel's presence in arthropods.     

Kinetic evidence for a dual cation role for muscle pyruvate kinase

The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg²⁺ and varying concentrations of Mn²⁺ or of Co²⁺. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co²⁺ and varying Mn²⁺. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn²⁺ is a better activator of the enzyme than is Mg²⁺, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni²⁺ or Zn²⁺ with Mg²⁺ suggests that Ni²⁺ and Zn²⁺ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg²⁺. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.     

Physical and kinetic properties of sheep liver pyridoxine kinase

Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn²⁺ > Mn²⁺ > Mg²⁺. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn²⁺ > Zn²⁺ > Mg²⁺. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K⁺, then Rb⁺ and NH₄⁺, whereas the enzyme had very little activity with Na⁺, Li⁺, or Cs⁺. Na⁺ did not interfere with the activation by K⁺. The activation of the kinase by K⁺, NH₄⁺, and Rb⁺ followed Michaelis-Menten kinetics, and the apparent K_m values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent K_m value of the enzyme for pyridoxine and had little or no effect on the K_m for ZnATP^2? or the V of the kinase-catalyzed reaction.     

Ion association reactions with biological membranes,studied with the fluorescent dye 1-anilino-8-naphthalenesulfonate

Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs⁺ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs⁺>Rb⁺>K⁺>Na⁺>Li⁺. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm ^–1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.     

Discrimination between monovalent and divalent cations by hydrophobic solvent-saturated membranes containing fixed negative charges

Summary Cellulose acetate-nitrate filters were saturated with hydrophobic solvent and interposed between various aqueous solutions. The membranes thus formed are cation permselective. The discrimination between a monovalent cation such as K⁺ and the alkaline earth group divalent cations is very sharp. The discrimination ratio is at least a few thousand times in favor of the monovalent cation. A major part of this discrimination is caused by the very low mobility of the divalent cation within the membrane compared with that of the monovalent cation. The remainder of the discrimination is caused by the selectivity of the membranes which prefer monovalent to divalent cations. There is a clear discrepancy between Ba⁺⁺ diffusibility and mobility within, the membrane. This implies that Ba⁺⁺ may move within the hydrophobic membrane as a neutral complex. Some similarity with natural biological membranes is indicated.     

Regeneration and inhibition of proton pumping activity of bacteriorhodopsin blue membrane by cationic amine anesthetics

Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca²⁺, Mg²⁺ and Mn²⁺) and monovalent metal cations (Li⁺, Na⁺, K⁺ and Cs⁺), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.     

The uptake and extrusion of monovalent cations by isolated heart mitochondria

Summary The factors involved in the movement of monovalent cations across the inner membrane of the isolated heart mitochondrion are reviewed. The evidence suggests that the energy-dependent uptake of K⁺ and Na⁺ which results in swelling of the matrix is an electrophoretic response to a negative internal potential. There are no clear cut indications that this electrophoretic cation movement is carrier-mediated and possible modes of entry which do not require a carrier are examined. The evidence also suggests that the monovalent cation for proton exchanger (Na⁺ > K⁺) present in the membrane may participate in the energy-dependent extrusion of accumulated ions. The two processes, electrophoretic cation uptake (swelling) and exchange-dependent cation extrusion (contraction) may represent a means of controlling the volume of the mitochondrion within the functioning cell. A number of indications point to the possibility that the volume control process may be mediated by the divalent cations Ca⁺² and Mg⁺². Studies with mercurial reagents also implicate certain membrane thiol groups in the postulated volume control process.An invited article.     

The Staphylococcal Pore-forming Leukotoxins Open Ca2+ Channels in the Membrane of Human Polymorphonuclear Neutrophils

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na⁺, K⁺, ethidium⁺) and is blocked by Ca²⁺ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium. Received: 8 May 1997/Revised: 22 December 1997     

Non-covalent (iso)guanosine-based ionophores for alkali(ne earth) cations

Different (iso)guanosine-based self-assembled ionophores give distinctly different results in extraction experiments with alkali(ne earth) cations. A lipophilic guanosine derivative gives good extraction results for K⁺, Rb⁺, Ca²⁺, Sr²⁺, and Ba²⁺ and in competition experiments it clearly favors the divalent Sr²⁺ (and Ba²⁺) cations. 1,3-Alternate calix[4]arene tetraguanosine hardly shows any improvement in the extraction percentages compared to its reference compound 1,3-alternate calix[4]arene tetraamide. This indicates that one G-quartet does not provide efficient cation complexation under these conditions. In the case of the lipophilic isoguanosine derivative there is a cation size dependent affinity for the monovalent cations (Cs⁺ ? Rb⁺ ? K⁺), but not for the divalent cations (Ca²⁺ > Ba²⁺ > Sr²⁺ > Mg²⁺). In competition experiments the isoguanosine derivative, unlike guanosine, does not discriminate between monovalent and divalent cations, giving an almost equal extraction of Cs⁺ and Ba²⁺.     

On the mechanism of anion uptake by plant roots

Summary The effect of the valence of the associated cation on Cl^–-uptake by excised barley roots grown in CaSO₄ has been studied at 26°, 6° and 2°C. The uptake of Cl^– relative to that of the associated cation was found to increase in the order: trivalent > divalent > monovalent. This was explained on the expected effect of the cation on the negative charge and potential of root surfaces. A lyotropic order was observed in case of monovalent cations, whereas divalent cations showed no such order. The order observed in Cl^–-uptake from chloride solutions of monovalent cations is associated with the ability of the absorbed cation to remove Ca and Mg from the roots. Li⁺ behaved similar to divalent cations in affecting the relative Cl^–-uptake from LiCl.As to the effect of temperature on the uptake of Cl^– and associated cation, it appears that Cl^– is not taken up to any great extent at 2°C whereas cations are still adsorbed at this low temperature. This has been explained on the assumption of the presence of negative adsorption spots on the root surface which can hold cations but not anions. It appears that Cl^–-uptake by roots requires the expenditure of energy to overcome repulsion arising from the negative surface.This work is supported by AEC contract AT (11-1) — 34 project 55.     

Extracellular Ca2+ controls outward rectification by apical cation channels in toad urinary bladder: Patch-clamp and whole-bladder studies

Summary Outward rectifying. cation channels were observed in the epithelial cells of the urinary bladder of the toad.Bufo marinus. As studied in isolated cells using the patch-clamp technique, the channel has an average conductance of 24 and 157 pS for pipette potentials between 0 and +60 mV and –60 to –100 mV, respectively, when the major cation in both bath and pipette solutions is K⁺. The conductance of the cannel decreasen with increasing dehydration energy of the permeant monovalent cation in the oder Rb⁺=K⁺>Na⁺>Li⁺. Reversal potentials near zero under biionic conditions imply that the permeabilities for all four of these cations are smiliar. The channel is sensitive to quinidine sulfate but not to amiloride. It shares several pharmacological and biophysical properties with an outwardly-rectifying, vasopressin-sensitive pical K⁺ conductive pathway described previously for the toad urinary bladder. We demonstrate, in both single-channel and whole-bladder studies, that the outward rectification is a consequence of interaction of the chanel with extracellular divalent cations, particularly Ca²⁺, which blocks inward but not outward current. Various divalent cations impart different degrees of outward rectification to the conductive pathway. Concentrations of Mg²⁺ and Ca²⁺ required for halfmaximal effect are 3×10^–4 and 10^–4 m, resopectively. For Co²⁺ the values are 10^–6 m at +50 mV and a 10^–4 m at +200 mV. The mechanism of blockade by divalent cations is not established, but does not seem to involve a voltage-dependent interaction in which the blocker penetrates the transmembrane electric field. In the absence of divalent cations in the mucosal solution, the magnitudes of inward current carried by Rb⁺, K⁺, Na⁺ and Li⁺ through the apical K⁺ pathway at any transepithelial voltage, are in the same order as in the single-channel studies. We propose that the cation channel observed by us in isolated epithelial cells is the single-channel correlate of the vasopressin-sensitive apical K⁺ conductive pathway in the toad urinary bladder and is also related to the oxytocin- and divalent cation-sensitive apical condictivity observed in frog skin and urinary bladder.     

Properties of pyruvate kinase from soybean nodule cytosol

The properties of pyruvate kinase from soybean (Glycine max L.) nodule cytosol were examined to determine what influence the N₂ fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K⁺ and NH₄⁺ concentrations. In the presence of physiological concentrations of K⁺ and high phosphoenolpyruvate (PEP) concentrations, NH₄⁺ inhibited enzyme activity. Comparisons of kinetic parameters (V_max and apparent K_a) for NH₄⁺ and K⁺ with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent K_a (monovalent cation) and K_m (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kinases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH₄⁺.     

Temperature dependent conformational changes at the active site of pyruvate kinase. A li nuclear magnetic resonance study.

The interaction of Li⁺, a weak activator of pyruvate kinase, with substrate and inhibitor complexes of the enzyme has been investigated by magnetic resonance techniques. Proton relaxation rate (PRR) titrations indicate that the dissociation constant of Li⁺ from the ternary enzyme-Mn(II)-phosphoenolpyruvate (P-enolpyruvate) complex is 15 mm at 5 °C and 17 mm at 30 °C. The electron paramagnetic resonance spectrum of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex is the superposition of spectra for two distinct species (Reed, G. H., and Cohn, M. (1973) J. Biol. Chem.248, 6436–6442). Low temperatures favor the form giving rise to the more nearly isotropic spectrum, whereas high temperatures favor the species giving rise to the anisotropic “K⁺-like” spectrum. ⁷Li nuclear magnetic resonance data are consistent with a model in which the two forms observed by epr correspond to differing Mn(II) to Li(I) distances. The form giving rise to the anisotropic spectrum is characterized by a Mn(II) to Li(I) distance of 4.7 Å, and in the more isotropic form this distance is approximately 9 Å. The 4.7 Å separation of the Mn(II) and Li(I) in the anisotropic form of the complex compares favorably with the 4.9 Å separation of Mn(II) and T1(I) (Reuben, J., and Kayne, F. J. (1971) J. Biol. Chem.246, 6227–6234) in the P-enolpyruvate complex, although T1⁺ is a much better activator of the pyruvate kinase reaction. Thus, a change in the distance between the monovalent and divalent cations does not account quantitatively for the lower activation by Li⁺, inasmuch as more than 50% of the enzyme-Mn(II)-Li(I)-P-enolpyruvate complex has the “active” conformation with respect to the separation of the cations and the epr spectrum of the complex. As reported previously (Reed, G. H., and Morgan, S. D. (1974) Biochemistry13, 3537–3541), the dissociation constant of oxalate and the epr spectrum for the ternary complex of pyruvate kinase with Mn(II) and oxalate are not influenced by the species of monovalent cation present. The nuclear relaxation rates of Li⁺ are increased in the presence of the ternary oxalate complex, although the separation of the Mn(II) and Li(I) appears to be much greater than for the “anisotropic” form of the P-enolpyruvate complex.     

The concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

The effect of monovalent and divalent cations on the activity of Streptococcus lactis C10 pyruvate kinase.

The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation.     

Changes in Conformation,Stability and Condensation of DNA by Univalent and Divalent Cations in Methanol-Water Mixtures

Abstract

Circular dichroism spectroscopy, absorption spectroscopy, measurements of T_m values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na⁺ or Cs⁺) concentration and also in the presence of divalent cations Ca²⁺, Mg²⁺, and Mn²⁺. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The T_m values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs⁺ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Salinomycin: a new monovalent cation ionophore.

The cation discriminations of salinomycin and its derivatives have been studied by measuring complexability with cations and transport rate of them across organic phase. Salinomycin exhibited a great preference for K⁺ over other monovalent and divalent cations in migrating cations into organic phase in two phase systems. The antibiotic mediated the transport of Na⁺ and Rb⁺ as effectively as that of K⁺ across CCl₄ bulk phase, but not those of Cs⁺, Mg²⁺, Ca²⁺, Sr²⁺. From the above results, salinomycin is concluded to act as an alkali ion carrier. The OH-acylated salinomycins retained the activity of parent compound, but the COOH-esterified salinomycins lost the activity.     

Availability of monovalent and divalent cations within intact chloroplasts for the action of ionophores nigericin and A23187

1. A23187 will uncouple electron transport by broken chloroplasts in a divalent cation dependent manner provided that they have been treated with a low concentration of EDTA.2. A23187 stimulates oxaloacetate-dependent oxygen evolution and inhibits phosphoglycerate reduction by intact chloroplasts isolated in a cation-free medium whereas the full effect of nigericin was dependent on the presence of external K⁺.3. Uncoupling of oxaloacetate reduction by A23187 in intact chloroplasts is inhibited by EDTA and this effect is overcome by excess Mg²⁺.4. The results suggest that divalent and not monovalent cations are available for collapsing the light-induced H⁺ gradient within the intact organelle.     

Some properties of pyruvate kinase extracted from Lycopersicon esculentum

Pyruvate kinase was extracted from Me₂CO-dried tissue of various parts of tomato plants. Recovery of the enzyme was improved by the inclusion of thiols in the extraction medium, and its stability was increased considerably in the presence of glycerol and to a lesser extent tetramethylammonium chloride. A phosphatase was present in the tissue extracts which hydrolyses phosphoenolpyruvate in the absence of added ADP. ATP inhibited pyruvate kinase but stimulated the phosphatase, while Mg²⁺ stimulated both enzymes. Data obtained suggest that tomato leaf pyruvate kinase has an absolute dependence on monovalent cations for activity, K⁺ being the principal activator. The phosphatase was inhibited non-selectively by monovalent cations. The total activity of pyruvate kinase and its concentration on a tissue fresh weight basis was greatest in the leaves, activity increasing with the maturity of the tissue. Less enzyme was present in roots, and least in the fruit.     

Effeet of Metal Ions on Activity of Exopectic Acid Transeliminase of Erwinia sp.

Contrary to the exopectic acid transeliminase of Clostridium multifermentans, that of Erwinia sp. was activated strongly by Na⁺ and to a much less extent by Ca²⁺. K⁺ had a small stimulating effect on the enzyme activity. Mn²⁺ and Co²⁺, like Ca²⁺, activated the enzyme weakly. Ba²⁺ and Mg²⁺ showed no and a slight inhibitory effect, respectively, on the activity.

An almost total loss of activity was caused by the addition of EDTA to the reaction mixture. In the presence of Na⁺ the enzyme activity was restored by addition of divalent cations. Individual monovalent cations or each of the divalent cations was ineffective in restoring the activity.