Altered adenosine triphosphatase activities of natural actomyosin from rat hearts perfused with isoprenaline and ouabain

Perfused rat hearts were treated with isoprenaline (10^?6M) or ouabain (5.5 × 10^?6M). The phosphate contents of troponin-I and myosin P light chains were established by radiolabelling with ³²P; in the case of the light chains, direct chemical analysis of total and of specifically alkali-labile phosphate was also performed. Addition of isoprenaline caused phosphorylation of both troponin-I and myosin P light chains, reaching a maximum increment, after several minutes, of 1 mol/mol and 0.30 mol/mol, respectively. The Mg²⁺-ATPase activities, at saturating Ca²⁺ concentrations, of natural actomyosin isolated from treated hearts were significantly depressed, and an inverse correlation was established between the phosphate content of troponin-I and the V_max[Ca²⁺] of this ATPase activity. The Ca²⁺ sensitivity of the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ was also decreased. These changes were all reversed by an incubation permitting dephosphorylation of proteins by endogenous phosphatases.Treatment of hearts with ouabain caused no increment in troponin-I phosphorylation, but increased the P light chain phosphate content to a maximum of 0.30 mol/mol after some minutes. A positive correlation was evident between phosphate content of the light chains (in all experiments) and the maximum myosin Ca²⁺-ATPase activities. In addition, the V_max[ATP] of the $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ of natural actomyosin was increased when light chain phosphorylation had occurred in the absence of troponin-I phosphorylation. P-light chain phosphorylation did not affect the Ca²⁺ sensitivity of $\text{Ca}{}^{\mathrm{2+}}\text{Mg}{}^{\mathrm{2+}}\text{-ATPase}$ activity.We suggest that the effects of phosphorylation of troponin-I are to diminish thin filament sensitivity to Ca²⁺, and to decrease the efficiency of the transduction process along neighbouring actin monomers, such that the number of actin-myosin crossbridge interactions is decreased even in the presence of Ca²⁺ excess. Phosphorylation of P light chains of myosin has an activating effect on myosin Ca²⁺-ATPase activity, as well as on the rate of cross-bridge formation.     

The effects of environmental factors on growth and the chemical and biochemical composition of marine diatoms. I. Light and temperature effects

Temperature and light interact to modify the chemical and biochemical composition of a nitrogen-limited marine diatom, Thalassiosira allenii Takano, grown at a constant dilution rate in continuous culture and under a light:dark cycle.The percent of the total ¹⁴C incorporated into protein, polysaccharide and lipid, the N/C ratio and the C/cell varied with temperature in a markedly non-linear manner. The N/cell was negatively correlated to temperature. The Chl $\text{a}\text{C}$ ratio was positively correlated with temperature under saturating light and non-saturating light for temperatures > 25 °C, but was constant under non-saturating light conditions for temperatures < 25 °C.Productivity index (PI) was negatively correlated to temperature under saturating light conditions, but did not vary under low light. In each case, the variation in PI with temperature was governed by the variation in Chl $\text{a}\text{C}$.The dark carbon loss rate was exponentially related to temperature and independent of light. Variation in the percent of the total ¹⁴C incorporated into protein and polysaccharide, the $\text{N}\text{C}$ ratio and C/cell was primarily due to the effects of N-limitation < 20 °C and primarily due to the effects of temperature > 20 °C. Variation in N/cell was primarily due to the effects of temperature over the entire range of temperature studied. Variation in Chl $\text{a}\text{C}$ was caused by the interaction of temperature and light effects.In most cases, temperature and nutrient effects interacted to govern how a particular parameter varied with temperature while light affected the range of values over which the parameter varied.The percent of the total ¹⁴C incorporated into protein exhibited a significant linear relationship with $\text{N}\text{C}$.The dark carbon loss rate, $\text{N}\text{C}$ ratio and Chl $\text{a}\text{C}$ ratio data were used to test the applicability of a model for the physiological adaptation of unicellular algae. The model, with parameters derived from a non-linear least-squares fit of the dark carbon loss rate data, adequately described the $\text{N}\text{C}$ ratio between 15 and 25 °C at 290 and 137 μE · m^?2 · s^?1, but failed to describe the data at 28 °C and at 48 μE · m^?2 · s^?1. The Chl $\text{a}\text{C}$ ratio was adequately described by the model under all light and temperature conditions.     

Calmodulin-like activity in the soluble fraction of Escherichia coli

A heat-stable factor with properties similar to those of calmodulin was found in the fraction containing Ca²⁺-dependent cyclic AMP phosphodiesterase of $\text{Escherichia}\text{coli}$. The factor activated such enzymes as cyclic nucleotide phosphodiesterase of bovine brain, (Ca²⁺,Mg²⁺)ATPase of human erythrocyte menbrane and myosin light chain kinase of rabbit myometrium in a Ca²⁺-dependent fashion with an apparent K_a of 5 × 10^?5$\text{M}$. The factor and brain calmodulin had no effect on the phosphodiesterase of $\text{E.}\text{coli}$. It may be concluded that calmodulin or a calmodulin-like protein occurs in prokaryotes.     

Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Effect of calcium and F-actin on the rate of SH2 modification in skeletal myosin

The rate constant of modification of a specific thiol group, SH₂, with N-ethylmaleimide (NEM) has been used to estimate the conformational change in the local area containing SH₂ (SH₂ region) of skeletal myosin as a structural probe. The rate of Mg²⁺-ATP-induced SH₂ modification of subfragment-1 (S-l) isozymes was regulated by Ca²⁺ in the pCa range below 6.4 and was not regulated in the pCa range above 6.4. No substantial difference between S-1 containing alkali light chain, A1, (S-1(A1)) and S-1 containing alkali light chain, A2, (S-1(A2)) was observed in the Ca²⁺-dependent rate of SH₂ modification. Due to the presence of this Ca²⁺ regulation in myosin (absence in S-1 isozymes) in the pCa range above 6.4, absence of 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) light chain in S-1 isozymes, and high affinity of Ca²⁺ for DTNB light chain, this Ca²⁺ regulation in the pCa range above 6.4 is possibly related to the Ca²⁺ binding to DTNB light chain. F-Actin, which is entirely free from tropomyosin and troponin, enhanced the rate of Mg²⁺-ATP-induced SH₂ modification of S-1 isozymes equally and of myosin, and reduced the Ca²⁺ sensitivity with an increase in F-actin concentration.     

Adenosine triphosphate sulfurylase from Penicillium chrysogenum equilibrium binding, substrate hydrolysis, and isotope exchange studies.

ATP sulfurylase from Penicillium chrysogenum was purified to homogeneity. The enzyme binds 8 mol of free ATP (K_s = 0.53 mM) or AMP (K_s = 0.50 mM) per 440,000 g. The results are consistent with our earlier report that the enzyme is composed of eight identical subunits of M_r 55,000 (J. W. Tweedie and I. H. Segel, 1971, Prep. Biochem. 1, 91–117; J. Biol. Chem. 246, 2438–2446). In the absence of cosubstrates, the purified enzyme catalyzes the hydrolysis of MgATP (to AMP and MgPP_i) and adenosine 5′-phosphosulfate (APS) (to AMP and SO₄^2?). MgATP hydrolysis is inhibited by nonreactive sulfate analogs such as nitrate, chlorate, and formate (uncompetitive with MgATP). In spite of the hydrolytic reactions it is possible to observe the binding of MgATP and APS to the enzyme in a qualitative (nonequilibrium) manner. Neither inorganic sulfate (the cosubstrate of the forward reaction) nor formate or inorganic phosphate (inhibitors competitive with sulfate) will bind to the free enzyme in detectable amounts in the absence or in the presence of Mg²⁺, Ca²⁺, free ATP, or a nonreactive analog of MgATP such as Mg-α,β-methylene-ATP. Similarly, inorganic pyrophosphate (the cosubstrate of the reverse reaction) will not bind in the absence or in the presence of Mg²⁺ or Ca²⁺. The induced binding of ³²P_i (presumably to the sulfate site) can be observed in the presence of MgATP. The results are consistent with the obligately ordered binding sequence deduced from the steady-state kinetics (J. Farley et al., 1976, J. Biol. Chem. 251, 4389–4397) and suggest that the subsites for SO^2?₄ or MgPP_i appear only after nucleotide cleavage to form E~AMP · MgPP_i or E~AMP · SO₄ complexes. The suggestion is supported by the relative values of K_ia (ca. 1 mm for MgATP) and K_iq (ca. 1 αm for APS) and by the inconsistent value of k_?1 calculated from $\text{V}{}_{\mathrm{<mtext>f</mtext>}}\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>a</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>A</mtext>}}$ (The value is considerably less than V_r) Purified ATP sulfurylase will also catalyze a Mg³²PP_i-MgATP exchange in the absence of SO₄^2?. A ³⁵SO₄^2?-APS exchange could not be demonstrated in the absence or presence of MgPP_i. This result was not unexpected: The rate of APS hydrolysis (or conversion to MgATP) is extremely rapid compared to the expected exchange rate. Also, the pool of APS at equilibrium is extremely small compared to the sulfate pool. The V values for molybdolysis, APS hydrolysis (in the absence of PP_i), ATP synthesis (from APS + MgPP_i), and Mg³²PP_i-MgATP exchange at saturating sulfate are all about equal (12–19 μmol × min^?1 × mg of enzyme^?1). The rates of Mg³²PP_i-MgATP exchange in the absence of sulfate, APS synthesis (from MgATP + sulfate), and MgATP hydrolysis (in the absence of sulfate) are considerably slower (0.10 – 0.35 μmol × min^?1 × mg of enzyme^?1). These results and the fact that k₄ calculated from $\text{V}{}_{\mathrm{<mtext>r</mtext>}}\text{K}{}_{\mathrm{<mtext>i</mtext><mtext>q</mtext>}}\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>Q</mtext>}}$ is considerably larger than V_f suggest that the rate-limiting step in the overall forward reaction is the isomerization reaction E~AMP-SO^2?₄ → EAPS. In the reverse direction the rate-limiting step may be SO^2?₄ release or isomerization of the E~AMP · MgPP_i · SO₄^2? complex. (The reaction appears to be rapid equilibrium ordered.) Reactions involving the synthesis or cleavage of APS are specific for Mg²⁺. Reactions involving the synthesis or cleavage of ATP will proceed with Mg²⁺, with Mn²⁺, and, at a lower rate, with Co²⁺. The results suggest that the enzyme possesses a Mg²⁺-preferring divalent cation (activator) binding site that is involved in APS synthesis and cleavage and is distinct from the MeATP or MePP_i site. The equilibrium binding of about one atom of ⁴⁵Ca²⁺ per subunit (possibly to the activator site) could be demonstrated (K_s = 1.4 mM).     

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.     

Weak acid-induced release of liposome-encapsulated carboxyfluorescein

Leakage of the entrapped anionic fluorophore carboxyfluorescein was used as a measure of the permeability of liposomes to several different acids. Carboxyfluorescein leakage increased with increasing buffer concentration at a given pH and depended on its chemical nature: apolar weak acids such as acetic or pyruvic acids induced fast leakage at relatively high pH (4 to 5), while glycine, aspartic, citric and hydrochloric acids induced leakage only at lower pH. Fluorescence leakage measurements reflected the acidification of the liposomes' aqueous spaces, which was primarily caused by the diffusion of undissociated acid molecules across the lipid bilayer. A simple mathematical model in accord with this hypothesis and assuming that carboxyfluorescein leakage was directly related to the proportion of its neutral lactone form, described satisfactorily the carboxyfluorescein leakage kinetics and allowed rough estimation of permeability coefficients for carboxyfluorescein (neutral lactone form; 9 · 10^?9 cm · s^?1), acetic acid ($\text{>1 · 10}{}^{\mathrm{?7}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$) and glycine (cation: 6 · 10^?9 cm · s^?1). These results are consistent with low effective proton permeability of liposomes ($\text{<5 · 10}{}^{\mathrm{?12}}\text{cm}\text{·}\text{s}{}^{\mathrm{?1}}$) and with the permeability coefficient of HCl (3 · 10^?3 cm · s^?1) reported by Nozaki and Tanford ((1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4324–4328). Diffusion of weak acid molecules across lipid membranes has implications for drug encapsulation and delivery, and may be of biological significance.     

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].     

The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin

Evidence is presented that both myosin and actomyosin in presence of Mg²⁺ and KCl catalyze an incorporation of ³²P_i into ATP. The rate with actomyosin is about $\text{1}\text{500}$ the rate of ATP hydrolysis; the rate with myosin is less than $\text{1}\text{100}$ of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of ³²P_i incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [³²P]-ATP present and the rate constant, k_?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔG^o for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔG^o for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage.     

The yield of chlorophyll a fluorescence as a means to test the various deexcitation mechanisms in the antenna system of green plants

With the aid of measurements of the fluorescence yield, the efficiency of the various deexcitation mechanisms of an exciton in the light-harvesting system has been determined. For this purpose, the fluorescence of dark-adapted as well as of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated and preilluminated leaves of Zea mays L. was excited by single ultrashort laser pulses of different energies. The experimental results have served for the fitting of solutions of rate equations, which describe the deexcitation by linear relaxation processes like fluorescence and radiationless transitions, by annihiation of excitons, and by traps both in the ground state and in an excited state. We have obtained the following results: a ratio of antenna chlorophyll molecules to Photosystem II traps of 600:1, an annihilation constant γ = 2·10^?8 cm³·s^?1, a mean trapping time of $\text{t}\text{?}\text{=0.5}\text{ns}$, a trapping probability for traps in the ground state of 2·10^?8 cm³·s^?1, and 6·10^?9 cm³·s^?1 for traps in an excited state.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

A multifunctional cyclic nucleotide- and Ca2+-independent protein kinase from rabbit skeletal muscle

A cyclic nucleotide- and Ca²⁺-independent protein kinase, initially identified as a glycogen synthase kinase (Itarte, E. and Huang, K.-P. (1979) J. Biol. Chem. $\text{254}$, 4052–4057), was also found to phosphorylate phosphorylase kinase and troponin from skeletal muscle as well as myosin light chain and myosin light chain kinase from both smooth and skeletal muscles. With the exception of myosin light chain from skeletal muscle, all the above-mentioned proteins are also substrates for the multifunctional cAMP-dependent protein kinase. The results suggest that this cyclic nucleotide- and Ca²⁺-independent protein kinase, like cAMP-dependent protein kinase, may have multiple cellular functions.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of $\text{3-O-}\text{methylglucose}$ in white fat cells was measured under equilibrium exchange conditions at $\text{3-O-}\text{methylglucose}$ concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10^?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the $\text{3-O-}\text{methylglucose}$ permeability was about 2 · 10^?9 cm · s^?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to $\text{3-O-}\text{methylglucose}$ at 37°C and in the presence of insulin was 4.3 · 10^?6 cm · s^?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of $\text{14.8 ± 0.44}\text{kcal/mol}$ was found. The corresponding value was $\text{13.9 ± 0.89}$ in the absence of insulin. The half saturating concentration of $\text{3-O-}\text{methylglucose}$ was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s^?1 per 1 intracellular water at 37°C.     

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.     

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

The kinetics of the Quin 2-Ca²⁺ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca²⁺ complex with a large excess of EGTA, EDTA or MgCl₂ resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s^?1 for the dissociation rate constant (k_?) at pH 7.2, 37°C, ± 3mM Mg²⁺. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k₊ was estimated to be ≥10⁹ M^?1s^?1, in agreement with the calculation from $\text{k}{}_{\mathrm{+}}\text{=}\text{k}{}_{\mathrm{?}}\text{K}$. These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca²⁺ transients.