Uptake of (-) 3H-norepinephrine by storage vesicles prepared from whole rat brain: properties of the uptake system and its inhibition by drugs.

Neurotransmitter storage vesicles were isolated from rat brain by differential centrifugation and the uptake of (?) ³H-norepinephrine was determined $\text{in vitro}$. Uptake showed a marked temperature dependence, an absolute requirement for ATP-Mg²⁺, and was inhibited $\text{in vitro}$ by reserpine. Uptake was linear for 5 min at 30°, but not at 37°. The uptake was saturable and displayed a single Km value of 4 × 10^?7 M. Other phenylamines and indoleamines displayed competitive inhibition of norepinephrine uptake; the affinities followed the rank order: reserpine>harmaline>serotonin>epinephrine> dopamine>norepinephrine>metaraminol. Uptake was reduced in vesicles isolated from rats treated intracisternally with 6-hydroxydopamine but not from rats treated with 5,6-dihydroxytryptamine, suggesting that most of the uptake occurs in catecholaminergic, and not serotonergic, vesicles. This method provides a ready characterization of pharmacologic effects on rat brain storage vesicle properties, as demonstrated by the prompt and complete inhibition of uptake $\text{in vitro}$ after administration of reserpine $\text{in vivo}$.     

A study of the sulfhydryl groups of rat brain hexokinase

Rat brain hexokinase (ATP: d-hexose-6-phosphotransferase, EC 2.7.1.1) is rapidly inactivated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). The inactivation follows monophasic first-order kinetics in either the absence of ligands (k = 0.641 min^?1 at 25 °C) or in the presence of saturating levels of ATP (free or complexed with Mg²⁺) or P₁; the inactivation rate is slightly increased ($\text{k ? 0.7}$ min ^?1) in the presence of ATP or P₁. In contrast, glucose and glucose-6-P markedly decrease the inactivation rate; inactivation in the presence of these ligands is biphasic, with two first-order rates ($\text{k ? 0.5}$ min^?1 and 0.01 min^?1) being distinguishable.The enzyme contains 14 sulfhydryl groups which react with 5,5′-dithiobis-(2-nitrobenzoate); reaction of these groups in the native enzyme is complete after 2 hr at 25 °C, or in approx 5 min with the urea or guanidine-denatured enzyme. In the native enzyme, three classes of sulfhydryl groups are distinguishable and are designated as F-, I-, or S-type based on their fast (k ? 0.7 min^?1), intermediate ($\text{k ? 0.5-0.7}$ min^?1), or slow ($\text{k ? 0.02}$ min^?1 rates of reaction with 5,5′-dithiobis-(2-nitrobenzoate). The correlation of inactivation rates with the rates for reaction of the I-type sulfhydryls indicates that the I-type sulfhydryls include residues necessary for catalytic activity. The F-type residues are clearly not required for activity.The effects of ATP, P₁, glucose, and glucose-6-P on the reactivity of the sulfhydryls have been determined. As in the absence of ligands, S-, I-, and F-type sulfhydryls could be distinguished. In the presence of saturating concentrations of these ligands, the F, I, and S classes of sulfhydryls contained respectively: with ATP, 1, 4, and 7 residues; with P₁, 1, 3, and 7 residues; with glucose, 1, 2, and 5 residues; with glucose-6-P, 1, 2, and 1 residues. Comparison with rate constants for inactivation in the presence of these ligands again indicated that I-type sulfhydryls were particularly important in maintenance of enzyme activity. The present results indicate considerable similarity between the reactivity of the sulfhydryl residues in rat brain hexokinase and the sulfhydryls of the bovine brain enzyme [V. D. Redkar and U. W. Kenkare (1972), J. Biol. Chem., 247, 7576–7584].     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Small-intestinal Na+/d-glucose cotransport. Inactivation of sugar transport and phlorizin binding by thiol-group and amino-group reagents

It has previously been shown that mercurials acting from the cytoplasmic side or from within the hydrophobic part of the membrane inactivate the small intestinal Na⁺/d-glucose cotransporter by blocking essential SH-groups (Klip, A., Grinstein, S. and Semenza, G. (1979) Biochim. Biophys. Acta 558, 233–245). Another (set of) sulfhydryl(s) which are critical for phlorizin binding and sugar transport function and which may lie on the luminal side of the brush border membrane, can be blocked by DTNB and 4,4′-dithiopyridine but not by $\text{N-}\text{ethylmaleimide}$. In addition, modification of amino groups by fluorescamine, reductive methylation and (under certain conditions) DIDS also lead to inactivation of the carrier's binding and transport functions. No evidence was obtained that any of the above groups is directly involved in the binding of either Na⁺/d-glucose or phlorizin, since none of these compounds prevented inactivation of the cotransporter.     

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation

Frozen aqueous suspensions of partially purified membrane-bound renal (Na⁺ + K⁺)-ATPase have been irradiated at –135°C with high-energy electrons. (Na⁺ + K⁺)-ATPase and K⁺-phosphatase activities are inactivated exponentially with apparent target sizes of $\text{184 ± 4 kDa and 125 ± 3 kDa}$, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na⁺ + K⁺)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na⁺?K⁺ exchange, $\text{201 ± 4}\text{kDa}$; (ATP + P_i)-activated Rb⁺?Rb⁺ exchange, $\text{206 ± 7}\text{kDa}$ and ATP-independent Rb⁺?Rb⁺ exchange, $\text{117 ± 4}\text{kDa}$. The apparent size of the α-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the β-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na⁺ + K⁺)-ATPase is αβ. (2) The inactivation target size for (Na⁺ + K⁺)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na⁺ and K⁺. (3) The target sizes, for K⁺-phosphatase (125 kDa) and ATP-independent Rb⁺?Rb⁺ exchange (117 kDa) are indistinguishable from that of the α-chain itself, suggesting that cation binding sites and transport pathways, and the $\text{p-}\text{nitrophenyl}$ phosphate binding site are located exclusively on the α-chain. (4) ATP-dependent activities appear to depend on the integrity of an αβ complex.     

Inactivation of cyclic GMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethylketone

Cyclic GMP-dependent protein kinase from bovine lung and cyclic AMP-dependent protein kinase from bovine heart are inactivated by N^α-tosyl-L-lysine chloromethylketone (TLCK) in the presence of cyclic GMP and cyclic AMP, respectively. The inactivation of both protein kinases is pseudo-first order, suggesting the rate limiting step is beyond the binding of TLCK. Cyclic GMP-dependent protein kinase is inactivated less than $\text{1}\text{4}$ as rapidly as cyclic AMP-dependent protein kinase, although it shows a higher apparent affinity for TLCK. Cyclic AMP stimulated the rate of inactivation of cyclic AMP-dependent protein kinase 10-fold but cyclic GMP stimulated the rate of inactivation of cyclic GMP-dependent protein kinase only 1.5-fold. The rate of inactivation of cyclic GMP-dependent protein kinase by TLCK is sufficiently rapid (half-time of about 30 min at 37°C with 2 mM TLCK) to account for the effects of TLCK on cell growth observed by others.     

cAMP-dependent protein kinase from Mucor rouxii: physical evidence of a ternary complex holoenzyme-cAMP

Gel electrophoresis and sucrose density gradient centrifugation techniques permitted the visualization for the first time of the ternary complex formed by the binding of cAMP to $\text{Mucor rouxii}$ cAMP-dependent protein kinase holoenzyme. The addition of 0.5 M NaCl or histone plus ATP-Mg⁺⁺, together with cAMP, dissociates the holoenzyme into free regulatory (R) and catalytic (C) subunits. At 4°C, cAMP bound to the holoenzyme is readily exchangeable with unlabeled cAMP (half life 2.5 min), while the nucleotide bound to the R subunit has a very slow exchange rate (half life 210 min). The amount of cAMP bound to R subunit is approximately twice the amount bound to holoenzyme at saturation.     

Role of substrates in Sr2+-induced oscillations of ionic fluxes in rat liver mitochondria

(1) The reason for substrate specificity of Sr²⁺-induced oscillating cation fluxes in isolated rat liver mitochondria was investigated. (2) With succinate as substrate, rotenone prevented oscillation. In this case the mitochondria were only partially able to take up added Sr²⁺ and did not take up any of the released K⁺. Addition of substances decreasing the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio (oxaloacetate or acetoacetate) restored the ability for reuptake of K⁺ and for complete uptake of Sr²⁺ and, therefore, oscillation. (3) Inhibition of substrate-level phosphorylation by arsenite or uncoupling of substrate-level phosphorylation by arsenate in the presence of oligomycin also suppressed the reuptake of cations. This effect of inhibition of substrate-level phosphorylation on oscillation could be circumvented by addition of ATP in the presence of oligomycin. (4) Prevention of the intramitochondrial regeneration of 2-oxoglutarate from acetyl-CoA and oxaloacetate by fluorocitrate or from endogenous glutamate by aminoxyacetate shortened the time during which oscillation with succinate as substrate could be observed. (5) From the key role of substrate level phosphorylation it is concluded that for the reuptake of K⁺ and Sr²⁺ during oscillation, sufficient GTP generation by the succinyl thiokinase (EC 6.2.1.4) reaction is essential. Therefore substrate level phosphorylation seems to be a necessary energy source additional to the respiratory chain. Since the latter process drives the active cation movements, the former may be required for the restoration of a sufficiently low proton conductance of the mitochondrial inner membrane. Oscillation in the absence of exogenous ATP therefore demands 2-oxoglutarate as substrate or the intramitochondrial generation of 2-oxoglutarate for the maintenance of a sufficient GTP production for a longer time.     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10^?2 min^?1. This provides a $\text{t}\text{1}\text{2}$ of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10^?2 min^?1 and 7 × 10^?3 min^?1 for uncatalyzed autoxidation and 4 × 10^?2 min^?1 and 1.1 × 10^?2 min^?1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.     

31P NMR lineshapes of β-P (ATP) in the presence of mg2+ and ca2+ : estimate of exchange rates

The ³¹ P NMR chemical shift of β-P of adenosine triphosphate (ATP) undergoes a substantial change (2&#x0303;–3 ppm) upon chelation of divalent ions such as Mg²⁺ or Ca²⁺. In the presence of nonsaturating amounts of Mg²⁺ or Ca²⁺, the lineshape of this resonance depends on the characteristic association and dissociation rates of these metal-ATP complexes. A procedure for computer simulation of this lineshape is outlined. A comparison of computer-simulated lineshapes with the experimental lineshapes obtained at 121 MHz was used to determine the following dissociation rate of Mg²⁺ and Ca²⁺ from their ATP complexes at 20°C and pH 8.0: Ca²⁺, > 3 × 10⁵ s^?1 (Hepes buffer); Mg²⁺, 1200 s^-1 (no buffer), 1000 s^-1 (Tris buffer) and 2100 s^?1 (Hepes buffer). The limits of error are ± 10% in these values. For the Mg²⁺ complexes, the rates were determined as a function of temperature to obtain activation energies (with a maximum deviation of 10% in the least-squares fit): 8.1 $\text{Kcal}\text{mole}$ (no buffer and Hepes buffer) and 6.8 $\text{kcal}\text{mole}$ (Tris buffer). Lineshapes of the β-Presonance simulated as a function of Mg²⁺ concentration, using 2100 s^?1 for the dissociation rate, are also presented. The computer simulation of lineshapes offers a reliable and straightforward method for the determination of exchange rates of diamagnetic cations from their ATP complexes, under a variety of sample conditions.     

Electrically induced Ca2+ transport across the membrane of Paramecium caudatum measured by means of flow-through technique

Transmembrane calcium fluxes related to excitation were studied in Paramecium caudatum. Radioactive (⁴⁵Ca²⁺) or inactive solution was flowed through a dense suspension of unlabelled or labelled cells, and radioactivity was monitored in the solution. The organisms were electrically stimulated by means of extracellular electrodes. As a result of stimulation Ca²⁺ uptake and release was measured. The uptake response dropped with increasing number of successive stimulation periods and increased with growing stimulus amplitude and duration. Maximum uptake was obtained at 20 V/cm and at least 60 s duration and for temperatures between 10 and 15°C. A Ca²⁺ influx of 700 pmol/1000 cells upon 1 min stimulation was measured at 15°C. This corresponds to an increment of the intraciliary [Ca²⁺] of about $\text{5 · 10}{}^{-4}\text{M}$. Ca²⁺ release was dependent on the stimulus amplitude in a similar manner as was Ca²⁺ uptake. Photographic recordings of the swimming behaviour of the organisms were used to interpret the flux data. At temperatures up to 15°C the cells swam backward perpendicular to the applied electric field of 10 to 20 V/cm. At 25°C they showed forward spiralling movement. For the first time evidence was brought for stimulated Ca²⁺ influx in Paramecium at physiological temperatures. It is concluded from the results that a strong active Ca²⁺ extrusion from the intraciliary space counteracts the influx. The Ca²⁺ pump rate must be at least $\text{8 · 10}{}^{12}$ calcium ions per s per cm² ciliary surface.     

Studies on (K+ + H+)-ATPase IV. Effects of phospholipase C treatment

(1) The total phospholipid content of a gradient purified ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase and K⁺ stimulated $\text{p-}\text{nitrophenylphosphatase}$ activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for ATP and temperature dependence of the gastric ($\text{K}{}^{\mathrm{+}}\text{+ H}{}^{\mathrm{+}}$)-ATPase, but slightly decreases the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ value for K⁺. (5) Phospholipase C treatment lowers the $\text{Ado}\text{PP[}\text{NH}\text{]P}$ binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

Kinetics of repair of O6-methylguanine in DNA by O6-methylguanine-DNA methyltransferase in, vitro and in, vivo

The demethylation of O⁶-methylguanine in double stranded DNA catalyzed by rat liver O⁶-methylguanine-DNA transmethylase was found to proceed much more rapidly when the DNA substrate was methylated to a high extent. When the content of O⁶-methylguanine in DNA was equal to 1 in 2000 guanines, the reaction was 90% complete within 2 min, but when the content was 1 in 500,000 it required 27 min at 37°C. These results suggest that the repair protein either moves along the DNA substrate or else has little selectivity for binding specifically to the sites containing O⁶-methylguanine rather than to the normal DNA. The repair of O⁶-methylguanine in rat liver $\text{in}\text{,}\text{vivo}$ occurred at rates comparable to those seen $\text{in}\text{,}\text{vitro}$ with the substrates alkylated to low extents and was virtually complete within 3 hours. These results provide strong evidence that this protein is the factor responsible for O⁶-methylguanine removal $\text{in}\text{,}\text{vivo}$ and explain the wide variation in time courses reported in the literature since substrates methylated to greatly different extents have been used for such experiments.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.