Organ-related distribution of phospholemman in the spiny dogfish Squalus acanthias.

The distribution of phospholemman among nine different organs of the spiny dogfish (Squalus acanthias) has been determined on the basis of Western blotting of microsomal material. Only rectal gland (100%), brain (43%), heart (18%), and kidney (19%) (abundancies as percent of the concentration in rectal gland) contained the protein, but not gill and colon. The relative abundance in the brain makes this organ a preferential test system for phospholemman in fishes that lack a rectal gland like teleosts.

Rate limitation of the Na(+),K(+)-ATPase pump cycle.

The kinetics of Na(+)-dependent phosphorylation of the Na(+),K(+)-ATPase by ATP were investigated via the stopped-flow technique using the fluorescent label RH421 (saturating [ATP], [Na(+)], and [Mg(2+)], pH 7.4, and 24 degrees C). The well-established effect of buffer composition on the E(2)-E(1) equilibrium was used as a tool to investigate the effect of the initial enzyme conformation on the rate of phosphorylation of the enzyme. Preincubation of pig kidney enzyme in 25 mM histidine and 0.1 mM EDTA solution (conditions favoring E(2)) yielded a 1/tau value of 59 s(-1). Addition of MgCl(2) (5 mM), NaCl (2 mM), or ATP (2 mM) to the preincubation solution resulted in increases in 1/tau to values of 129, 167, and 143 s(-1), respectively. The increases can be attributed to a shift in the enzyme conformational equilibrium before phosphorylation from the E(2) state to an E(1) or E(1)-like state. The results thus demonstrate conclusively that the E(2) --> E(1) transition does in fact limit the rate of subsequent reactions of the pump cycle. Based on the experimental results, the rate constant of the E(2) --> E(1) transition under physiological conditions could be estimated to be approximately 65 s(-1) for pig kidney enzyme and 90 s(-1) for enzyme from rabbit kidney. Taking into account the rates of other partial reactions, computer simulations show these values to be consistent with the turnover number of the enzyme cycle (approximately 48 s(-1) and approximately 43 s(-1) for pig and rabbit, respectively) calculated from steady-state measurements. For enzyme of the alpha(1) isoform the E(2) --> E(1) conformational change is thus shown to be the major rate-determining step of the entire enzyme cycle.

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.

Concentration jump experiments for the precise determination of rate constants of reverse reactions in the millisecond time range.

Low dissociation or reverse rate constants of single-step or multistep complex formation equilibria are usually obtained with reduced precision from standard stopped-flow binding experiments by determination of the intercept of the concentration dependence of k(obs). Large and fast concentration jumps, based on two different step-motor-driven mixing setups, are performed with 60-300-fold dilutions that allow the precise, convenient, and independent determination of dissociation rate constants in the range of approximately 0.1-100 s(-1) in a single stopped-flow dissociation experiment. A theoretical basis is developed for the design and for the evaluation of such dilution experiments by considering the rebinding occurring during dissociation. The kinetics of three chemical systems are investigated, the binding of Mg2+ to 8-hydroxyquinoline as well as of Ca2+ and K+ to the cryptand [2.2.2], by carrying out standard stopped-flow binding as well as dissociation experiments employing various dilution factors. The advantage of the dilution method for investigating chemical and biological systems is emphasized.

Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle.

Dephosphorylation kinetics of pig kidney Na+,K+-ATPase.

The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be 190 s-1).

Functional immobilization of biomembrane fragments on planar waveguides for the investigation of side-directed ligand binding by surface-confined fluorescence.

A method for the functional immobilization of Na,K-ATPase-rich membrane fragments on planar metal oxide waveguides has been developed. A novel optical technique based on the highly sensitive detection of surface-confined fluorescence in the evanescent field of the waveguide allowed us to investigate the interactions of the immobilized protein with cations and ligands. For specific binding studies, a FITC-Na,K-ATPase was used, which had been labelled covalently within the ATP-binding domain of the protein. Fluorophore labels of the surface-bound enzyme can be selectively excited in the evanescent field. A preserved functional activity of the immobilized enzyme was only found when a phospholipid monolayer was preassembled onto the hydrophobic chip surface to form a gentle, biocompatible interface. In situ atomic force microscopy (AFM) was used to examine and optimize the conditions for the lipid and membrane fragment assembly and the quality of the formed layers. The enzyme's functional activity was tested by selective K+ cation binding, interaction with anti-fluorescein antibody 4-4-20, phosphorylation of the protein and binding of inhibitory ligand ouabain. The comparison with corresponding fluorescence intensity changes found in bulk solution provides information about the side-directed surface binding of the Na,K-ATPase membrane fragments. The affinity constants of K+ ions to the Na,K-ATPase was the same for the immobilized and the non-immobilized enzyme, providing evidence for the highly native environment on the surface. The method for the functional immobilization of membrane fragments on waveguide surfaces will be the basis for future applications in pharmaceutical research where advanced methods for exploring the molecular mechanisms of membrane receptor targets and drug screening are required.

Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.

The kinetics of Na+-dependent partial reactions of the Na+,K+-ATPase were investigated via the stopped-flow technique using the fluorescent labels RH421 and BIPM. After the enzyme is mixed with MgATP, both labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau1 approximately 180 s-1 (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3 --> E2P(Na+)3 + ADP). The rate of the phosphorylation reaction measured by the acid quenched-flow technique was 190 s-1 at 100 microM ATP, suggesting that phosphorylation controls the kinetics of the RH421 signal and that the conformational change is very fast (>/=600 s-1). The rate of the RH421 signal was optimal at pH 7.5. The Na+ concentration dependence of 1/tau1 showed half-saturation at a Na+ concentration of 8-10 mM with positive cooperativity involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high affinity ATP binding site determined from the ATP concentration dependence of 1/tau1 was 7.0 (+/-0.6) microM, while the apparent Kd for the low affinity site and the rate constant for the E2 to E1 conformational change evaluated in the absence of Mg2+ were 143 (+/-17) microM and

High-pressure stopped-flow spectrometer for kinetic studies of fast reactions by absorbance and fluorescence detection.

The development of a stopped-flow instrument that operates over a temperature range of -40 to +100 °C and up to 200 MPa is described. The system has been designed so that measurements can be performed in absorbance and fluorescence modes simultaneously, without dismantling the unit. It can easily be combined with an optical system of a conventional ambient pressure setup by using light guides. Optimum optical performance and a wide operating wavelength range (220-850 nm) are achieved as the light is not passing through the pressurizing fluid. A special design for the pistons has been developed; thus, the apparatus has proven to be leak-free, even under extreme conditions (high pressure, low temperature, various solvents). The dead time of the system is found to be less than 2 ms at 298 K and is pressure independent up to 200 MPa. We examined the kinetics for the formation of the Mg(2+)-8-hydroxyquinoline chelate in aqueous solutions at pH 8.0 in order to develop a convenient alternative test method for high-pressure stopped-flow spectrometers with absorption and fluorescence detection.

Time-resolved response of fluorescent alkali ion indicators and detection of short-lived intermediates upon binding to molecular cavities.

Stopped-flow kinetic studies have been performed to determine the kinetic parameters of K(+) binding to the fluorescent cryptand F222 and of Na(+)binding to F221 at pH 8.O. The results clearly indicate that a comparatively stable intermediate is formed before the rate-limiting binding step occurs with a rate constant around 30 s(-1) under the chosen experimental conditions. The conversion of the intermediate to the final cation complex is assigned to the final penetration of the already bound, but still partially solvated cation into the ligand's cavity. The main fluorescence intensity change found upon cation binding is attributed to the second reaction step, and not to the fast, initial binding reaction. The comparatively slow overall binding reaction is interpreted on the bases of a special solvate substitution mechanism which, in principle, can also account for the 1500 times slower binding of Ca 2(+) to F221. With regard to time-resolved analytical Na(+) and K(+) determinations, the response times under the chosen conditions are around 20 ms. Differentiation between Na(+) and Ca(2+), for example, is possible with F221 on the basis of completely different response times.

Conformational changes of Na,K-ATPase probed with eosin Y.

Time-resolved fluorescence and binding studies have been carried out on Na,K-ATPase in the presence of the fluorescent dye eosin Y to obtain thermodynamic and kinetic parameters for the interaction of the enzyme with different cations. Eosin Y binding is indicated by a 3 ns fluorescence decay process and is observed only in the presence of mono- and divalent cations. This type of cation binding is interpreted as a nonselective electrostatic interaction, with negatively charged groups of the enzyme providing a high-affinity eosin Y binding site. Eosin Y binding is observed only under conditions where the enzyme exists in the conformational state F1. The kinetic parameters of eosin Y binding have been determined employing stopped-flow fluorometry.

scratch, a pan-neural gene encoding a zinc finger protein related to snail, promotes neuronal development.

The Drosophila scratch (scrt) gene is expressed in most or all neuronal precursor cells and encodes a predicted zinc finger transcription factor closely related to the product of the mesoderm determination gene snail (sna). Adult flies homozygous for scrt null alleles have a reduced number of photoreceptors in the eye, and embryos lacking the function of both scrt and the pan-neural gene deadpan (dpn), which encodes a basic helix-loop-helix (bHLH) protein, exhibit a significant loss of neurons. Conversely, ectopic expression of a scrt transgene during embryonic and adult development leads to the production of supernumerary neurons. Consistent with scrt functioning as a transcription factor, various genes are more broadly expressed than normal in scrt null mutants. Reciprocally, these same genes are expressed at reduced levels in response to ectopic scrt expression. We propose that scrt promotes neuronal cell fates by suppressing expression of genes promoting non-neuronal cell fates. We discuss the similarities between the roles of the ancestrally related scrt, sna, and escargot (esc) genes in regulating cell fate choices.

Electric parameters of Na+/K(+)-ATPase by measurements of the fluorescence-detected electric dichroism.

The electric parameters of Na+/K(+)-ATPase labeled by FITC have been characterized by measurements of the fluorescence-detected electric dichroism. The fluorescence emission was measured with polarizers at the magic angle and the light for excitation was usually polarized parallel to the field vector. The FITC-Na+/K(+)-ATPase preparations exhibit a negative electric dichroism at field strengths up to about 600 V/cm and a positive dichroism at higher field strengths. Pulse reversal experiments reveal a dominant permanent electric moment at low electric field strengths and an increasing contribution from an induced electric moment at higher field strengths. The dichroism rise curves and the transients upon pulse reversal show two relaxation processes with opposite amplitudes, whereas the dichroism decay curves in most cases can be represented by single exponentials at a reasonable accuracy. The amplitude A2 associated with the slower of the rise processes is dominant at low field strengths and also approaches saturation already at low field strengths. The dependence of A2 on the electric field strength is consistent with the orientation function for permanent dipoles and cannot be represented by the orientation function for induced dipoles. The fitted permanent dipole moment is in the range of 3.5 x 10(-24) Cm [1 x 10(6) D] and shows only a relatively small decrease with increasing ionic strength. The stationary values of the electric dichroism up to field strengths E < or = 800 V/cm can be represented with high accuracy by an orientation function for disk-shaped particles with a permanent moment along the particle symmetry axis and an induced moment along the semi-major axis. The permanent electric moment determined according to this function is consistent with the one obtained from the amplitudes A2. In summary, our measurements indicate that Na+/K(+)-ATPase is associated with a large permanent electric moment directed perpendicular to the membrane plane. The dipole moment per ATPase monomer unit is estimated to be 1.4 x 10(-27) Cm [430 +/- 50 D].

High-performance liquid chromatographic purification of extremely hydrophobic peptides: transmembrane segments.

Transmembrane peptides of integral membrane proteins often exhibit extremely high hydrophobicity. Therefore, the solubility of such peptides in solvents commonly used in HPLC is usually very low and the interaction with generally applied stationary phases such as silica gel or C18 reversed phases appears to be extremely strong, which makes the characterization and purification of these peptides difficult. The analytical characterization and preparative separation of the synthesized M1 transmembrane sequence of the inhibitory glycine receptor M(r) 48,000 subunit and some of its fragments is shown. M1 and its larger fragments could be dissolved in a dichloromethane-hexafluoro-2-propanol mixture containing a trace amount of pyridine for their separation on a C4 phase by employing linear two-component gradients of formic acid-2-propanol and formic acid-water with ratios up to 4:1 (v/v). Conditions to avoid formylation of the peptides are indicated.

Atonal is the proneural gene for Drosophila photoreceptors.

The Drosophila peripheral nervous system comprises four major types of sensory element: external sense organs (such as mechano-sensory bristles), chordotonal organs (internal stretch receptors), multiple dendritic neurons, and photoreceptors. During development, the selection of neural precursors for external sense organs requires the proneural genes of the achaete-scute complex, which encode basic-helix-loop-helix transcription factors. These genes do not, however, control precursor selection for chordotonal organs or photoreceptors, raising the question of whether other proneural genes exist or a different mechanism of neurogenesis operates. Here we show that atonal (ato), originally isolated as a proneural gene for chordotonal organs, is also the proneural gene for photoreceptors. Pattern formation in the Drosophila eye involves a succession of cell fate specifications. Of the eight photoreceptors within each ommatidium of the compound eye, the photoreceptor R8 is the first to appear in the eye imaginal disc, right behind the morphogenetic furrow. The appearance of other photoreceptors (R1-7) follows in a defined sequence that is thought to arise by induction from R8 (refs 8, 9, 11, 12). We find that photoreceptor formation requires the function of atonal at the morphogenetic furrow and that atonal is specifically required for R8 selection. Formation of other photoreceptors does not directly require atonal function, but does depend on R8 selection by atonal. Thus, photoreceptors are selected by two mechanisms: R8 by a proneural mechanism, and R1-7 by local recruitment.

Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase.

Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase. Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan. The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites. alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy. Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not. It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis. Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP. Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365. With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.

Tryptophan-free Escherichia coli F1-ATPase.

We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alpha W513F/gamma W108Y/gamma W206Y/beta W107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showed Vmax and Km both 65% higher than wild-type, resulting in kcat/Km the same as wild-type. The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type. Catalytic-site nucleotide-binding characteristics were measured using the analog lin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan. We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability and in vivo assembly.

Fluorescence studies and semiempirical calculations on alkali ion indicators.

Two newly synthesized cryptands act as sensitive Na(+)- and K(+)-selective indicators for cation concentrations above 20 µM. The fluorescence properties change markedly upon cation binding. In addition, the free ligands exhibit a pronounced sensitivity to pH, which is considerably lower for the cation complexes. Time resolved fluorescence is characterized by a decay time of about 5 ns that is attributed to the diprotonated protolytic state of the uncomplexed ligands. Semiempirical calculations show the systematic influence of the nitrogen lone pairs or the N-H bond on the stability of the system. The cause of the strong fluorescence intensity increase observed upon protonation of the fluorescent cryptands may be attributed to an increase in the S1-T x energy gap as a consequence of bridgehead nitrogen protonation.

lin-Benzo-ATP and-ADP: Versatile fluorescent probes for spectroscopic and biochemical studies.

lin-Benzo-adenine nucleotides can act not only as probes for fluorescence studies but also as structural active site probes for enzymes. To understand the basic properties oflin-benzo-ATP and-ADP, protolysis and Mg(2+) and Ca(2+), binding are investigated between pH 6.2 and pH 8.5 by spectrophotometric and spectrofluorometric titrations. Based on a reaction model, a set of equilibrium constants is determined which is consistent with all available experimental results. The pK values of the Mg(2+) and Ca(2+) complex oflin-benzo-ATP in the chosen medium are 4.6 and 4.1, respectively, and those for the corresponding diphosphate are 3.1 and 2.8, respectively. Fluorescence and absorption spectra are reported.