Temperature Dependences of Torque Generation and Membrane Voltage in the Bacterial Flagellar Motor

In their natural habitats bacteria are frequently exposed to sudden changes in temperature that have been shown to affect their swimming. With our believed to be new methods of rapid temperature control for single-molecule microscopy, we measured here the thermal response of the Na⁺-driven chimeric motor expressed in Escherichia coli cells. Motor torque at low load (0.35 μm bead) increased linearly with temperature, twofold between 15°C and 40°C, and torque at high load (1.0 μm bead) was independent of temperature, as reported for the H⁺-driven motor. Single cell membrane voltages were measured by fluorescence imaging and these were almost constant (∼120 mV) over the same temperature range. When the motor was heated above 40°C for 1–2 min the torque at high load dropped reversibly, recovering upon cooling below 40°C. This response was repeatable over as many as 10 heating cycles. Both increases and decreases in torque showed stepwise torque changes with unitary size ∼150 pN nm, close to the torque of a single stator at room temperature (∼180 pN nm), indicating that dynamic stator dissociation occurs at high temperature, with rebinding upon cooling. Our results suggest that the temperature-dependent assembly of stators is a general feature of flagellar motors.     

Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli

The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na⁺-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque-speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na⁺). With full expression of stator proteins the torque-speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at ∼ 2200 pN nm) from stall up to a “knee” speed of ∼ 420 Hz, and then falls linearly with speed, extrapolating to zero torque at ∼ 910 Hz. Motors containing one to five stators generated ∼ 200 pN nm per stator at speeds up to ∼ 100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to ∼ 400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to ∼ 1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque-speed relationship.     

Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na+-Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na⁺-driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na⁺-driven motor than in the MotA-FliG interactions in the H⁺-driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na⁺-driven motor seem to be more complex than those previously proposed in the H⁺-driven motor.     

Mutations targeting the C-terminal domain of FliG can disrupt motor assembly in the Na(+)-driven flagella of Vibrio alginolyticus

The torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor by an as yet unknown mechanism to engage the rotor-stator interactions. Here, we investigated stator assembly using a mutational approach of the Na⁺-driven polar flagellar motor of Vibrio alginolyticus, whose stator is localized at the flagellated cell pole. We mutated a rotor protein, FliG, which is located at the C ring of the basal body and closely participates in torque generation, and found that point mutation L259Q, L270R or L271P completely abolishes both motility and polar localization of the stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. Those residues are localized at the core of the globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, which suggests that those mutations induce quite large structural alterations at the interface responsible for the rotor-stator interaction. These results show that the C-terminal domain of FliG is critical for the proper assembly of PomA/PomB stator complexes around the rotor and probably functions as the target of the stator at the rotor side.     

Functional role of a conserved aspartic acid residue in the motor of the Na-driven flagellum from Vibrio cholerae

The flagellar motor consists of a rotor and a stator and couples the flux of cations (H⁺ or Na⁺) to the generation of the torque necessary to drive flagellum rotation. The inner membrane proteins PomA and PomB are stator components of the Na⁺-driven flagellar motor from Vibrio cholerae. Affinity-tagged variants of PomA and PomB were co-expressed in trans in the non-motile V. cholerae pomAB deletion strain to study the role of the conserved D23 in the transmembrane helix of PomB. At pH 9, the D23E variant restored motility to 100% of that observed with wild type PomB, whereas the D23N variant resulted in a non-motile phenotype, indicating that a carboxylic group at position 23 in PomB is important for flagellum rotation. Motility tests at decreasing pH revealed a pronounced decline of flagellar function with a motor complex containing the PomB-D23E variant. It is suggested that the protonation state of the glutamate residue at position 23 determines the performance of the flagellar motor by altering the affinity of Na⁺ to PomB. The conserved aspartate residue in the transmembrane helix of PomB and its H⁺-dependent homologs might act as a ligand for the coupling cation in the flagellar motor.     

Intragenic Suppressor of a Plug Deletion Nonmotility Mutation in PotB,a Chimeric Stator Protein of Sodium-Driven Flagella

The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H⁺ or Na⁺ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na⁺-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na⁺-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na⁺-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation.     

Temperature dependence of the rotation and hydrolysis activities of F1-ATPase

F₁-ATPase, a water-soluble portion of the enzyme ATP synthase, is a rotary molecular motor driven by ATP hydrolysis. To learn how the kinetics of rotation are regulated, we have investigated the rotational characteristics of a thermophilic F₁-ATPase over the temperature range 4-50°C by attaching a polystyrene bead (or bead duplex) to the rotor subunit and observing its rotation under a microscope. The apparent rate of ATP binding estimated at low ATP concentrations increased from 1.2 × 10⁶ M⁻¹ s⁻¹ at 4°C to 4.3 × 10⁷ M⁻¹ s⁻¹ at 40°C, whereas the torque estimated at 2 mM ATP remained around 40 pN·nm over 4-50°C. The rotation was stepwise at 4°C, even at the saturating ATP concentration of 2 mM, indicating the presence of a hitherto unresolved rate-limiting reaction that occurs at ATP-waiting angles. We also measured the ATP hydrolysis activity in bulk solution at 4-65°C. F₁-ATPase tends to be inactivated by binding ADP tightly. Both the inactivation and reactivation rates were found to rise sharply with temperature, and above 30°C, equilibrium between the active and inactive forms was reached within 2 s, the majority being inactive. Rapid inactivation at high temperatures is consistent with the physiological role of this enzyme, ATP synthesis, in the thermophile.     

Forced rotation of Na-driven flagellar motor in a coupling ion-free environment

Rotational characteristics of Na⁺-driven flagellar motor in the presence and absence of coupling ion were analyzed by electrorotation method. The motor rotated spontaneously in the presence of Na⁺, and the rotation accelerated or decelerated following the direction of the applied external torque. The spontaneous motor rotation was inhibited by removal of external Na⁺, however, the motor could be forcibly rotated by relatively small external torque applied by the electrorotation apparatus. The observed characteristic of the motor was completely different from that of ATP-driven motor systems, which form rigor bond when their energy source, ATP, is absent. The internal resistance of the flagellar motor increased significantly when the coupling ion could not access the inside of the motor, suggesting that the interaction between the rotor and the stator is changed by the binding of the coupling ion to the internal sites of the motor.     

Force variability depends on core and muscle temperature

The aim of this study was to investigate if voluntary activation and force variability during maximal voluntary contraction (MVC) depends more on muscle (local) or body (core) temperature. Ten volunteers performed a 2-min MVC of the knee extensors under the control (CON) conditions (ambient temperature (21 °C), relative humidity (30%), and air velocity (∼0.1 m/s)) as well as after heating (HT) and cooling (CL) of the lower body. During water manipulation procedure lower body was immersed up to the waist in a water bath at ∼44 °C for 45 min for HT experiment, and ∼15 °C for 30 min for CL experiment. Peak torque, torque variability, muscle voluntary activation and half-relaxation time were assessed during the exercise. HT increased muscle (2.8±0.2 °C) and rectal (1.9±0.1 °C) temperatures while CL lowered muscle (2.2±0.2 °C) temperature, but did not affect rectal temperature. During 2-min MVC, peak torque decreased (P<0.05; SP>90%) and to a lower level in HT compared to CON and CL experiments (52.6±2.3% versus 69.0±2.3% and 65.6±1.9% MVC, respectively, P<0.05; SP>90%). Torque variability increased significantly during exercise and was significantly larger in HT and lower in CL compared to CON experiment. Voluntary activation of exercising muscle was more depressed in HT (i.e. greater central fatigue) and the smallest effect was found in CL compared to CON. In conclusion increased core and muscle temperature impairs voluntary activation and increases force variability of the exercising muscles while a local muscle cooling decrease force variability but has a small effect on central fatigue.     

Simple, inexpensive attainment and measurement of very high cooling and warming rates

We have developed a simple, inexpensive system (<$300 US) for measuring cooling and warming rates of small (∼ 0.1 μl) aqueous samples at rates as high as 10⁵ °C/min. The measurement system itself, can track rates approaching one million °C/min. For temperature sensing, a Type T thermocouple with 50 μm wire was used. The thermocouple output voltage was read with an inexpensive USB based digital oscilloscope interfaced to a laptop computer, and the raw data were processed with MS Excel.     

Water transport in epididymal and ejaculated rhesus monkey (Macaca mulatta) sperm during freezing

In the present study, we report the effects of cooling ejaculated and epididymal rhesus monkey (Macacamulatta) sperm with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal sperm cell suspensions were obtained at a cooling rate of 20 °C/min in the absence of any cryoprotective agents and in the presence of 0.7 M of glycerol, as well. Using previously published values, the macaque sperm cell was modeled as a cylinder of length 73.83 μm with a radius of 0.40 μm and an osmotically inactive cell volume, V_b, of 0.772V_o, where V_o is the isotonic cell volume. This translated to a surface area, SA to initial water volume, WV ratio of ∼22 μm⁻¹. By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the best-fit membrane permeability parameters (reference membrane permeability to water at 0 °C, L_pg or L_pg[cpa] and the activation energy, E_Lp or E_Lp[cpa]) were found to range from: L_pg or L_pg[cpa] = 0.0020-0.0029 μm/min-atm; E_Lp or E_Lp[cpa]) = 10.6-18.3 kcal/mole. By incorporating these membrane permeability parameters in a recently developed equation (optimal cooling rate, ; where the units of B_opt are °C/min, E_Lp or E_Lp[cpa] are kcal/mole, L_pg or L_pg[cpa] are μm/min-atm and SA/WV are μm⁻¹), we determined the optimal rates of freezing macaque sperm to be ∼23 °C/min (ejaculated sperm in the absence of CPAs), ∼29 °C/min (ejaculated sperm in the presence of glycerol), ∼24 °C/min (epididymal sperm in the absence of CPAs) and ∼24 °C/min (epididymal sperm in the presence of glycerol). In conclusion, the subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal macaque spermatozoa under corresponding cooling conditions.     

Evaluation of the Duty Ratio of the Bacterial Flagellar Motor by Dynamic Load Control

The bacterial flagellar motor is one of the most complex and sophisticated nanomachineries in nature. A duty ratio D is a fraction of time that the stator and the rotor interact and is a fundamental property to characterize the motor but remains to be determined. It is known that the stator units of the motor bind to and dissociate from the motor dynamically to control the motor torque depending on the load on the motor. At low load, at which the kinetics such as proton translocation speed limits the rotation rate, the dependency of the rotation rate on the number of stator units N implies D: the dependency becomes larger for smaller D. Contradicting observations supporting both the small and large D have been reported. A dilemma is that it is difficult to explore a broad range of N at low load because the stator units easily dissociate, and N is limited to one or two at vanishing load. Here, we develop an electrorotation method to dynamically control the load on the flagellar motor of Salmonella with a calibrated magnitude of the torque. By instantly reducing the load for keeping N high, we observed that the speed at low load depends on N, implying a small duty ratio. We recovered the torque-speed curves of individual motors and evaluated the duty ratio to be 0.14 ± 0.04 from the correlation between the torque at high load and the rotation rate at low load.     

Coiled-Coil Response to Mechanical Force: Global Stability and Local Cracking

Coiled coils are important structural motifs formed by two or more amphipathic α-helices that twist into a supercoil. These motifs are found in a wide range of proteins, including motor proteins and structural proteins, that are known to transmit mechanical loads. We analyze atomically detailed simulations of coiled-coil cracking under load with Milestoning. Milestoning is an approach that captures the main features of the process in a network, quantifying kinetics and thermodynamics. A 112-residue segment of the β-myosin S2 domain was subjected to constant-magnitude (0–200 pN) and constant-direction tensile forces in molecular dynamics simulations. Twenty 20 ns straightforward simulations at several load levels revealed that initial single-residue cracking events (Ψ > 90°) at loads <100 pN were accompanied by rapid refolding without either intra- or interhelix unfolding propagation. Only initial unfolding events at the highest load (200 pN) regularly propagated along and between helices. Analysis of hydrophobic interactions and of interhelix hydrogen bonds did not show significant variation as a function of load. Unfolding events were overwhelmingly located in the vicinity of E929, a charged residue in a hydrophobic position of the heptad repeat. Milestoning network analysis of E929 cracking determined that the mean first-passage time ranges from 20 ns (200 pN) to 80 ns (50 pN), which is ∼20 times the mean first-passage time of an isolated helix with the same sequence.     

Gelation of high-methoxy pectin by enzymic de-esterification in the presence of calcium ions: a preliminary evaluation

Cohesive gels have been obtained by de-esterification of 1.0 wt % high-methoxy citrus pectin (degree of esterification ≈ 68%) in the presence of Ca²⁺ cations, using a commercial preparation (NovoShape) of fungal methyl esterase cloned from Aspergillus aculeatus. A convenient rate of network formation (gelation within ∼30 min) was achieved at an enzyme concentration of 0.2 PEU/g pectin. At a Ca²⁺-concentration of 40 mM and incubation temperature of 20 °C, severe syneresis (>7% of sample mass) was observed, but release of fluid decreased with decreasing concentration of Ca²⁺ and increasing temperature of incubation, becoming undetectable for 10 mM Ca²⁺ at 30 °C. Under these conditions, progressive development of solid-like character (storage modulus, G′) was observed during 160 min of enzymic de-esterification, and the mechanical spectrum recorded at the end of the incubation period had the form typical of a biopolymer gel. On subsequent heating to 70 °C, dissociation of the gel network (sigmoidal reduction in G′ and G″) was observed. At or above the midpoint temperature of this melting process (∼50 °C), there was no indication of gel formation on enzymic de-esterification (at 50 or 60 °C). At lower temperatures (20, 30 and 40 °C), the rate of gelation (assessed visually) showed no systematic increase as the incubation temperature was increased towards the temperature-optimum of the enzyme (∼50 °C). This unexpected behaviour is attributed to competition between faster de-esterification and slower formation of Ca²⁺-induced ‘egg-box’ junctions.     

Neither helix in the coiled coil region of the axle of F1-ATPase plays a significant role in torque production

F₁-ATPase is an ATP-driven rotary molecular motor in which the central γ-subunit rotates inside the cylinder made of α₃β₃ subunits. The amino and carboxy termini of the γ-subunit form the axle, an α-helical coiled coil that deeply penetrates the stator cylinder. We previously truncated the axle step by step, starting with the longer carboxy terminus and then cutting both termini at the same levels, resulting in a slower yet considerably powerful rotation. Here we examine the role of each helix by truncating only the carboxy terminus by 25-40 amino-acid residues. Longer truncation impaired the stability of the motor complex severely: 40 deletions failed to yield rotating the complex. Up to 36 deletions, however, the mutants produced an apparent torque at nearly half of the wild-type torque, independent of truncation length. Time-averaged rotary speeds were low because of load-dependent stumbling at 120° intervals, even with saturating ATP. Comparison with our previous work indicates that half the normal torque is produced at the orifice of the stator. The very tip of the carboxy terminus adds the other half, whereas neither helix in the middle of the axle contributes much to torque generation and the rapid progress of catalysis. None of the residues of the entire axle played a specific decisive role in rotation.     

Two flagellar stators and their roles in motility and virulence in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 6605

The motor proteins around the flagellar basal body consist of two cytoplasmic membrane proteins, MotA and MotB, and function as a complex that acts as the stator to generate the torque that drives rotation. Genome analysis of several Pseudomonas syringae pathovars revealed that there are two sets of genes encoding motor proteins: motAB and motCD. Deduced amino acid sequences for MotA/B and MotC/D showed homologies to the H⁺-driven stator from Escherichia coli and Na⁺-driven stator from Vibrio alginolyticus, respectively. However, the swimming motility of P. syringae pv. tabaci (Pta) 6605 was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but not by the sodium stator-specific inhibitor phenamil. To identify a gene encoding the stator protein required for motility, ∆motAB, ∆motCD, and ∆motABCD mutants were generated. The ∆motCD mutant had remarkably reduced and the ∆motABCD mutant completely abolished swimming motilities, whereas the ∆motAB mutant retained some degree of these abilities. The ∆motCD and ∆motABCD mutants did not produce N-acyl-homoserine lactones (AHLs), quorum-sensing molecules in this pathogen, and remarkably reduced the ability to cause disease in host tobacco leaves, as we previously observed in the ∆fliC mutant strain. These results strongly indicate that both stator pairs in Pta 6605 are proton-dependent and that MotCD is important for not only flagellar motility but also for production of AHLs and the ability to cause disease in host plants.     

The Effect of Myofilament Compliance on Kinetics of Force Generation by Myosin Motors in Muscle

We use the inhibitor of isometric force of skeletal muscle N-benzyl-p-toluene sulfonamide (BTS) to decrease, in a dose dependent way, the number of myosin motors attached to actin during the steady isometric contraction of single fibers from frog skeletal muscle (4°C, 2.1 μm sarcomere length). In this way we can reduce the strain in the myofilament compliance during the isometric tetanus (T₀) from 3.54 nm in the control solution (T_0,NR) to ∼0.5 nm in 1 μM BTS, where T₀ is reduced to ∼0.15 T_0,NR. The quick force recovery after a step release (1-3 nm per half-sarcomere) becomes faster with the increase of BTS concentration and the decrease of T₀. The simulation of quick force recovery with a multistate model of force generation, that adapts Huxley and Simmons model to account for both the high stiffness of the myosin motor (∼3 pN/nm) and the myofilament compliance, shows that the increase in the rate of quick force recovery by BTS is explained by the reduced strain in the myofilaments, consequent to the decrease in half-sarcomere force. The model estimates that i), for the same half-sarcomere release the state transition kinetics in the myosin motor are five times faster in the absence of filament compliance than in the control; and ii), the rate of force recovery from zero to T₀ is ∼6000/s in the absence of filament compliance.     

Effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea

The effects of temperature, salinity, and irradiance on the growth of the dinoflagellate Akashiwo sanguinea were examined in the laboratory. The irradiance at the light compensation point (I₀) was 14.40 μmol m^− 2 s^− 1 and the irradiance at growth saturation (I_s) was 114 μmol m^− 2 s^− 1. We exposed A. sanguinea to 48 combinations of temperature (5-30 °C) and salinity (5-40) under saturating irradiance; it exhibited its maximum growth rate of 1.13 divisions/day at a combination of 25 °C and salinity of 20. A. sanguinea was able to grow at temperatures from 10 to 30 °C and salinities from 10 to 40. This study revealed that A. sanguinea was a eurythermal and euryhaline organism; in Japan it should have formed blooms in early summer, when salinity was relatively low. In addition, it was noteworthy that A. sanguinea had markedly cold-durability, retaining the motile form of vegetative cells for more than 50 days at 5 °C and at salinities of 25-30.     

None of the Rotor Residues of F1-ATPase Are Essential for Torque Generation

F₁-ATPase is a powerful rotary molecular motor that can rotate an object several hundred times as large as the motor itself against the viscous friction of water. Forced reverse rotation has been shown to lead to ATP synthesis, implying that the mechanical work against the motor’s high torque can be converted into the chemical energy of ATP. The minimal composition of the motor protein is α₃β₃γ subunits, where the central rotor subunit γ turns inside a stator cylinder made of alternately arranged α₃β₃ subunits using the energy derived from ATP hydrolysis. The rotor consists of an axle, a coiled coil of the amino- and carboxyl-terminal α-helices of γ, which deeply penetrates the stator cylinder, and a globular protrusion that juts out from the stator. Previous work has shown that, for a thermophilic F₁, significant portions of the axle can be truncated and the motor still rotates a submicron sized bead duplex, indicating generation of up to half the wild-type (WT) torque. Here, we inquire if any specific interactions between the stator and the rest of the rotor are needed for the generation of a sizable torque. We truncated the protruding portion of the rotor and replaced part of the remaining axle residues such that every residue of the rotor has been deleted or replaced in this or previous truncation mutants. This protrusionless construct showed an unloaded rotary speed about a quarter of the WT, and generated one-third to one-half of the WT torque. No residue-specific interactions are needed for this much performance. F₁ is so designed that the basic rotor-stator interactions for torque generation and control of catalysis rely solely upon the shape and size of the rotor at very low resolution. Additional tailored interactions augment the torque to allow ATP synthesis under physiological conditions.     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature