Involvement of thyroid gland in the development of migratory disposition in the redheaded bunting, Emberiza bruniceps

In the redheaded bunting Emberiza bruniceps, thyroidectomy inhibited premigratory fattening and nocturnal restlessness—two characteristics of avian migration—observed in caged birds during the premigratory period (March/April). Thyroxine (T₄) and triiodothyronine (T₃) administration in thyroidectomized birds stimulated locomotor activity and restored the loss in body weight. Annual variations in circulating thyroid hormone concentrations revealed a significant rise in $\text{T}{}_3\text{T}{}_4$ ratio prior to spring migration in both years studied. This increase in circulating $\text{T}{}_3\text{T}{}_4$ ratio may be associated with the development of migratory disposition in this bird. There was no increase in circulating $\text{T}{}_3\text{T}{}_4$ ratio prior to autumnal migration, however, plasma T₄ increased significantly. Different thyroidal mechanisms are most likely involved in spring and fall migratory periods. While T₃ remained low throughout, apart from the characteristic spring rise, high T₄ levels in E. bruniceps were associated with periods of reproduction and molting, the latter coinciding partly with autumnal migration.     

Influence of duration of incubation on zooplankton respiration and excretion results

Respiration (O), ammonium (NH₄), phosphate (PO₄), total nitrogen (N_T) and phosphorus (P_T) excretions were measured on mixed zooplankton during 3-, 6-, 9-, 12-, 21-, and 24-h incubation periods at 20–23 C. The excretion rates of PO₄, N_T. and P_T decrease during a 21-h period, while rates of respiration and excretion of NH{IN4} are constant. The percentage of inorganic nitrogen excreted increases regularly from 3 h (30–40% of total nitrogen) to 21 h (70–80%) and it could be either due to a bacterial activity which was measured or to a decrease with time of organic nitrogen excreted because of starvation. $\text{O}\text{N}{}_{\mathrm{T}}$, $\text{O}\text{PO}{}_4$, $\text{O}\text{P}{}_{\mathrm{T}}$, and $\text{NH}{}_4\text{PO}{}_4$ ratios increase during the first 9 h of incubation; the percentage of inorganic phosphorus excreted is higher at the very beginning and then remains constant from 6 to 24 h. $\text{O}\text{NH}{}_4$ and $\text{N}{}_{\mathrm{T}}\text{P}{}_{\mathrm{T}}$ ratios are constant during a 24-h term, which makes them useful metabolic indexes.     

Binding of the structural protein soc to the head shell of bacteriophage T4

Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N⁴-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C₃), -A-U-G-A- (mutant C₄) and -A-U-A-A- (mutant $\text{C}{}_3\text{C}{}_4$) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C₃), 3.2 (C₄) and 0.3 ($\text{C}{}_3\text{C}{}_4$). The finding that mutant C₃ no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C₄ RNA bound more efficiently than wild type, and that mutant $\text{C}{}_3\text{C}{}_4$ RNA showed substantial ribosome binding capacity whereas the single mutant C₃ did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of $\text{C}{}_3\text{C}{}_4$ this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Additivity of isotope effects for successive deuteration in the deacetylation of acetyl-α-chymotrypsin

α-Chymotrypsin, converted to the acetyl enzyme by the p-nitrophenyl esters of CH₃COOH, CH₂DCOOH, CHD₂COOH, and CD₃COOH, undergoes deacetylation at pH 7.6 (phosphate buffer) and 25°C with secondary isotope effects of $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CH}{}_2\text{D}\text{)}\text{= 0.985 ± 0.006}$, $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CHD}{}_2\text{)}\text{= 0.971 ± 0.010}$, and $\text{k(}\text{CH}{}_3\text{)}\text{k(}\text{CD}{}_3\text{)}\text{= 0.956 ± 0.008}$. These isotope effects obey the simple additivity rule (“Rule of the Geometric Mean”) to within 20 J/mol, corresponding to about 5–6% of the maximum isotope effect for carbonyl addition. Thus, to this level, the three hydrogenic sites of the acetyl group are not rendered distinct in their contributions to the overall isotope effect even in the chiral environment of the chymotrypsin active site.     

Hydration of Escherichia coli lipids: Deuterium T1 relaxation time studies of phosphatidylglycerol,phosphatidylethanolamine and phosphatidylcholine

The hydration properties of Escherichia coli lipids (phosphatidylglycerol, phosphatidylethanolamine) and synthetic $\text{1,2-}\text{dioleoyl}\text{-sn-}\text{glycero-3-phosphocholine}$ in H₂O/²H₂O mixtures (9:1, v/v) were investigated with ²H-NMR. Comparison of the ²H₂O spin lattice relaxation time ($\text{T}{}_1$) as a function of the water content revealed a remarkable quantitative similarity of all three lipid-H₂O systems. Two distinct hydration regions could be discerned in the $\text{T}{}_1$ relaxation time profile. (1) A minimum of 11–16 water molecules was needed to form a primary hydration shell, characterized by an average relaxation time of $\text{T}{}_1\text{≈ 90}\text{ms}$. (2) Additional water was found to be in exchange with the primary hydration shell. The exchange process could be described in terms of a two-site exchange model, assuming rapid exchange between bulk water with $\text{T}{}_1\text{= 500}\text{ms}$ and hydration water with $\text{T}{}_1\text{= 80–120}\text{ms}$. Analysis of the linewidth and the residual quadrupole splitting (at low water content) confirmed the size of the primary hydration layer. However, each lipid-water system exhibited a somewhat different linewidth behavior, and a detailed molecular interpretation appeared to be preposterous.     

Preferential solvation of methylmercurated calf thymus deoxyribonucleic acid.

The changes in polymer-solvent interactions that occur when native calf thymus DNA is dialyzed against Na₂SO₄ solutions of a given ionic strength and buffer concentration but of varying concentrations in methylmercuric hydroxide have been investigated with the help of solution density measurements at 25 °C and pH 6.8–7.0. From measurements executed under equilibrium dialysis conditions at the three salt levels 5 mm, 0.05 m, and 0.5 m Na₂SO₄ (m refers to molality) and in the presence of 5 mm cacodylic acid buffer, the density increments $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ for native calf thymus DNA were determined as a function of CH₃HgOH concentration. $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ was found not to vary with organomercurial concentration, irrespective of the concentration of supporting electrolyte, until a certain CH₃HgOH concentration level has been reached, viz., , beyond which $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ increases strongly with increasing concentration of CH₃HgOH. As is shown by optical melting, $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ becomes a function of organomercurial concentration the moment DNA undergoes denaturation brought about by the complexing of CH₃HgOH with the various N-binding sites of the base residues in the DNA double helix.Polymer-solvent interactions, expressed in terms of preferential water interactions (“net hydration”) and preferential salt interactions (“salt solvation”), were derived from the $\text{(}\text{??}\text{?c}{}_2\text{)}{}_{\mathrm{\mu }}{}^0$ data in combination with data obtained on the preferential interaction of CH₃HgOH with denatured DNA and data on the partial specific volumes of all major solution components, gathered from density measurements on solutions with fixed concentrations of diffusible components. Evidence is presented which shows that denaturation in general decreases the net hydration while salt becomes preferentially associated with the polyelectrolyte. This process is further amplified by the interaction of CH₃HgOH with denatured DNA: Methylmercurated DNA alters the redistribution of diffusible components at dialysis equilibrium to such an extent that in a formal sense large amounts of water are rejected from the immediate vicinity of the polymer. The molecular implications of these findings are explored. The results are further discussed in the light of previous findings where the methylmercury-induced denaturation of DNA had been studied with the help of buoyant density measurements in a Cs₂SO₄ density gradient and by velocity-sedimentation in a variety of sulfate media.     

Proton nuclear magnetic resonance studies of the manganese (II) binding site of concanavalin A. Effect of saccharides on the solvent relaxation rates

The longitudinal relaxation rate ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$) of water protons was studied in solutions of Mn(II)-concanavalin A at a number of frequencies. These relaxation rates were lowered in the presence of a variety of saccharides which have affinities for concanavalin A which range over two orders of magnitude. A good correlation was found in which saccharides which bind tightly have the greatest effect and saccharides which bind weakly or not at all have little effect on the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ values. The temperature dependence of the proton relaxation rates showed that the lowering of these rates in the presence of saccharides was most likely due to a change in the exchange rate of solvent interacting with protein-bound Mn(II), $\text{1}\text{T}{}_{\mathrm{<mtext>m</mtext>}}$.An analysis of the temperature and frequency dependence of the $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ (transverse) solvent proton relaxation rates resulted in evaluation of a number of parameters for solvent water molecules interacting in the first coordination sphere of Mn(II) bound to concanavalin A. The ratio of the number of water molecules (q) to the Mn(II)-proton distance (r) obtained from a computer fit of the data over a limited temperature range is in accord with the findings of Koenig et al. ((1973) Proc. Nat. Acad. Sci.70, 475) and Meirovitch and Kalb ((1973) Biochim. Biophys. Acta303, 258). However, our studies of $\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$ of water over a more extensive temperature range are best fit with the following conclusions: at low temperatures (<20 °C), the data are consistent with an outer-sphere relaxation process. At higher temperatures (> 30 °C), the water molecule in the inner coordination sphere of the bound Mn(II) begins exchanging more rapidly and contributes to the relaxation processes ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>p</mtext>}}$ and $\text{1}\text{T}{}_{\mathrm{<mtext>2</mtext><mtext>p</mtext>}}$). The relaxation time of protons in the inner coordination shell, T_1M, contributes over the entire temperature range and produces a frequency dependence in the relaxivity data from 6 to 100 MH_z since the contributions to the correlation times are in the range 10^?9-10^?8 sec.     

Phase transition characteristics of diphosphatidylglycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers: Mono- and divalent metal ion effects

Synthesis and phase transition characteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature ($\text{T}{}_{\mathrm{<mtext>m</mtext>}}$) and the enthalpy of transition ($\text{ΔH}$) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric $\text{sn-3,3-}\text{and}\text{sn-1,1-}\text{phosphatidyldiacylglycerol}$ and for the diastereomeric, $\text{meso}\text{-sn-1,3-}\text{phosphatidyldiacylglycerol}$ are approximately the same. The molar enthalpy for the transition of cardiolipin-NH₄⁺ bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C_{1 6}-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e. the highest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Ca²⁺- cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca²⁺. The lowest $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ is observed for Rb⁺-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.     

Cellular aspects of tolerance. VII. Inheritance of the resistance to tolerance induction

The half-lives of elimination ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of ¹³¹I-RGG from the body of normal A or Balb/c animals was much longer than the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of SJL mice. At all ages, the $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of normal hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to or longer than that of the A or Balb/c parents. Thus, in terms of the $\text{T}\text{1}\text{2}$ of normal animals, the SJL responsiveness to ¹³¹I-RGG appeared to be a recessive trait. Tolerance could be induced in newborn animals and, in terms of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, the degree of unresponsiveness at the age of 6 weeks, was the same in A, Balb/c, A × SJL, and Balb/c × SJL animals but was much shorter in SJL mice. Thus, in neonatally induced tolerance, the duration of tolerance was recessive for the SJL type. The average $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ after tolerance induction in 3-week-old hybrids (A × SJL, SJL × A, Balb/c × SJL) was similar to that of the A or Balb/c parent, but by the 8th and 12th week it approached the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of the SJL parent. Comparing 8-week-old hybrids, the average $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ was longest in A × SJL hybrids and identical in SJL × A and Balb/c × SJL mice. An examination of $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ distribution in various 8- and 12-week-old crosses and backcrosses revealed a fairly large proportion of individuals with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ which was intermediate between SJL and the other parent. There was a tendency for this number to decrease in 12 weeks as compared to 8-week-old mice. In 8-week-old mice, the number of animals with intermediate $\text{T}{}_{\mathrm{<mtext>built1</mtext><mtext>2</mtext>}}$ was smallest when SJL was the maternal animal [(SJL × A); SJL × (A × SJL); SJL × (SJL × A)]. There was no link between $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of tolerant animals and either the immunoglobulin allotype (MuAl/MuA2) or the C5 eniotype (MuB1 positive/MuB1 negative).     

Binding of bovine cytochrome b5 to phosphatidycholine liposomes Characterization of the reconstituted lipid-protein vesicles

Cytochrome $\text{b}{}_5$ was extracted and purified from beef liver by a detergent method (cytochrome $\text{d-b}{}_5$). The hydrophilic moiety which carries the heme group (cytochrome $\text{t-b}{}_5$) was prepared by trypsin action upon pure cytochrome $\text{d-b}{}_5$.Single-shelled lecithin liposomes form complexes with cytochromes $\text{d-b}{}_5$ up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [³H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome $\text{t-b}{}_5$ does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature $\text{T}{}_{\mathrm{M}}$. i.e. when the aliphatic chains are in a crystalline state, and above $\text{T}{}_{\mathrm{M}}$, when the alipathic chain are in a fluid state.¹H NMR spectra show that even at the maximum cytochrome $\text{d-b}{}_5$ concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex.     

The characterization of energized and partially de-energized (respiration-independent) β-galactoside transport into escherichia coli

Unidirectional fluxes of [¹⁴C]lactose by whole cells of Escherichia coli under highly energized and partially de-energized (in the presence of CN^?) conditions are analyzed kinetically.When the cells are energized, the value for $\text{V}$ influx is $\text{0.45 ± 0.01}\text{mM}$ internal concentration increment/s and $\text{K}{}_{\mathrm{t}}$ is $\text{0.26 ± 0.03}\text{mM}$. At an external concentration of 0.61 mM the steady-state internal concentration is 0.25 M, reached after about 1h. The maximum steady-state concentration ratio is $\text{2 · 10}{}^3$.The efflux process under these conditions is non-saturable, being linearly dependent upon internal concentration over the range 25–250 mM with a first-order rate constant of $\text{8.8 ± 0.2 · 10}{}^{\mathrm{?4}}\text{s}{}^{\mathrm{?1}}$.The transport in the presence of CN^? is active, with a maximum concentration ratio (internal concentration/external concentration) of 104, and the uptake is mimicked by anoxia (< 70 ppm O₂).The effects of CN^? are to lower the $\text{V}$ for influx and to change the efflux from a non-saturable to a saturable process with a value for $\text{K}{}_{\mathrm{t}}$ (60 mM) intermediate between that for energized efflux ($\text{> 250}\text{mM}$) and influxe (0.3–0.6 mM), the latter value not changing appreciably. Partial de-energization thus affects both the influx and efflux processes.     

An improved method for determining breath H2 as an indicator of carbohydrate malabsorption

The hydrogen content of whole human breath (not concentrated or end-expiratory samples), useful as an index of carbohydrate malabsorption, can be determined accurately by gas chromatographic analysis using a thermal conductivity detector and, for the first time, and electronic peak integrator. Standard gas mixtures ($\text{H}{}_2\text{Ar}$ or preferably $\text{H}{}_2\text{N}{}_2$) give peak areas linearly related to hydrogen content. For repeatable results, it is necessary to pressurize samples in a 0.5-ml sample loop. Five minutes are required for the analysis of a sample with a printed report of the area of the H₂ peak (arbitrary units) and concentration of H₂ (parts per million). Standard samples can be stored in special impervious bags for up to 3 days without loss. Details of bag construction are given.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Amiloride stimulation of sodium transport in the presence of calcium and a divalent cation chelator

Amiloride in nM to μM concentrations stimulates the short circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) of the toad urinary bladder by as much as 120% when applied in conjunction with apical Ca²⁺ and a divalent cation chelator. A significant decrease in transepithelial resistance ($\text{R}{}_{\mathrm{<mtext>t</mtext>}}$) is observed simultaneously. This response is spontaneously reversible and its amplitude is dependent upon apical sodium concentrations. The stimulated $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ persisted when acetazolamide (1 mM) was introduced, HPO₄^2? substituted for HCO₃^? or SO₄^2? replaced Cl^?. Consequently, the increase in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is not due to the change of Cl^?, H⁺ or HCO₃^? flux. This behavior in a ‘tight’ epithelium may be related to the mechanism controlling apical sodium permeability.