Electron diffraction study on single crystals of l-type and dl-type lecithins

An electron diffraction study was carried out on thin single micro-crystals of l-type and dl-type dipalmitoyl lecithins grown in xylene suspensions and fine net patterns were obtained and the mechanism of the thermotropic phase transitions of them was clarified.From the apparent structure of diffraction patterns in low temperature, it is confirmed that the two dimensional lattices have p mm symmetry in l-type and in dl-type lecithins. Lattice parameters from the [001] projection are $\text{d}{}_{100}\text{= 9.9}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.8}\text{A}\text{?}$ in l-type, and $\text{d}{}_{100}\text{= 17.2}\text{A}\text{?}$ and $\text{d}{}_{010}\text{= 8.9}\text{A}\text{?}$ in dl-type.With anisotropic variation of dimensions along a and b axes, i.e. contraction for a and expansion for b, induced by temperature rise by electron irradiation during the observation, these diffraction patterns of the lattices of l-type and dl-type were transformed into those characterized by the six diffraction spots having nearly the same spacings. Four of them are observed on slightly outer and two are slightly inner positions as compared with their mean spacings of about (4.1 Å)^?1 in l-type and about (4.2 Å)^?1 in dl-type. The changes in the patterns observed indicate that at low temperatures the hydrocarbon chains are nearly perpendicular to the layer in dl-type lipid, and tilted with a more complicated packing in l-type ones. The dimension along a in dl-type is twice as large as that in l-type.     

Cytoskeletal proteins used as marker of differentiation in mouse terato;carcinoma cells

The cyclic AMP receptor protein (CRP) of Escherichia coli has been crystallized. The crystals are orthorhombic, space group $\text{P2}{}_1\text{2}{}_1\text{2}{}_1\text{, a = 46.5}\text{A}\text{?}$, b = 97.1 Å, $\text{c = 105.4}\text{A}\text{?}$, with one dimeric CRP molecule per asymmetric unit.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.     

Preliminary crystallographic data for actinidin,a thiol protease from Actinidia chinensis

Crystals of actinidin, a thiol protease from the fruit of Actinidia chinensis, which are suitable for high-resolution X-ray diffraction studies, have been obtained. The space-group is $\text{P2}{}_1\text{2}{}_1\text{2}{}_1\text{,}\text{with}\text{a = 78.1}\text{A}\text{?}\text{, 6 = 81.2}\text{A}\text{?}\text{and}\text{c = 33.0}\text{A}\text{?}$. The asymmetric unit contains one molecule, of molecular weight about 26,000.     

Can diffusion coefficients be estimated from plasma clearance curves of intact animals?

Certain substances, when injected intravenously, are removed from the plasma in accordance with the simple power law, C = At^?a. C is the concentration in plasma; A and a are constants. It is suggested that when a is of the order of 1·5, this power law may result from diffusion of the injected substance under conditions of spherical symmetry. It is shown for two such substances, that their diffusion coefficient in plasma, D, is given approximately by the expression $\text{D =}\text{1}\text{4}\text{(M}{}_{\mathrm{E}}\text{/A)}{}^{\mathrm{<mtext>2</mtext><mtext>3</mtext>}}$, where M_E is the effective diffusing mass.     

Preliminary x-ray crystallographic data for the semisynthetic non-covalent complex of residues 1 to 118 and 111 to 124 of bovine pancreatic ribonuclease

The enzymically active, semisynthetic complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease has been crystallized at pH 5.7 from $\text{(}\text{NH}{}_4\text{)}{}_2\text{SO}{}_4\text{CsCl}$ solutions. The crystals belong to space group P3₂21, have unit cell dimensions $\text{a}\text{and}\text{b = 64.4}\text{A}\text{?}\text{, c = 64.5}\text{A}\text{?}\text{,}\text{and}$ γ = 120° and are isomorphous with form M of ribonuclease A as well as forms W and R of ribonuclease S. They diffract well and may be expected to yield a structure defined to at least 3.0 Å resolution.     

Polar group interaction and molecular packing of membrane lipids: The crystal structure of lysophosphatidylethanolamine

The conformation and molecular packing of 3-palmitoyl-dl-glycerol-1-phosphoryl-ethanolamine has been determined by a single crystal analysis (R = 0.115); it crystallizes in the monoclinic space group $\text{P2}{}_1\text{a}$ with a unit cell of $\text{a = 7.66}\text{A}\text{?}\text{, b = 9.08}\text{A}\text{?}\text{, c = 37.08}\text{A}\text{?}\text{and}\text{β = 90.2 °}$, with four molecules per unit cell. The molecules exist as configurational and conformational enantiomers and pack in a bilayer arrangement. The phosphorylethanolamine groups have an orientation parallel to the layer surface. The hydrocarbon chains are arranged according to the T∥ chain packing mode and adopt an extreme tilt of 57.5 ° with respect to the layer normal. The free glycerol hydroxyl group forms an intramolecular hydrogen bond with, a phosphate oxygen and thus affects the conformation and orientation of the head group. The phosphorylethanolamine dipoles are oriented parallel to each other in double rows, while they are antiparallel and form a continuous network in dilauroylphosphatidylethanolamine (Elder et al., 1977). The area per molecule in 3-palmitoyl-dl-glycerol-1-phosphorylethanolamine (34.8 Ų) is less than in diacylphosphatidylethanolamine (38.6 Ų), indicating that in the latter the hydrocarbon chains determine the molecular cross-section. The significance of the interaction and space requirement of the phosphorylethanolamine group for the phase behaviour of phosphatidylethanolamine is discussed.     

On equivalent forms of Whittaker's similarity index

For two N-species assemblages A, B with specific proportionate abundances of the ith species a_i, b_l respectively, we consider the equality

$\text{∑}\text{t=1}\text{N}\text{c}{}_{\mathrm{i}}\text{= 1?}\text{1}\text{2}\text{∑}\text{t=1}\text{N}\text{|a}{}_{\mathrm{i}}\text{?b}{}_{\mathrm{i}}\text{|, c}{}_{\mathrm{i}}\text{=}\text{a}{}_{\mathrm{i}}\text{a}{}_{\mathrm{i}}\text{? b}{}_{\mathrm{i}}\text{b}{}_{\mathrm{i}}\text{a}{}_{\mathrm{i}}\text{> b}{}_{\mathrm{i}}\text{, 0?a,b,c?1}$

. The left-hand term is known as Sanders' minimum faunal abundance value, while the right side is referred to as Whittaker's similarity index. Both measures are commonly used in community studies. Equality between these two measures obtains only when proportionate abundances are utilized. We develop equivalent formulation which is valid for absolute abundance data, reduces to the Sanders-Whittaker equality when proportionate abundance data are employed, and is more sensitive to differences in species abundance distributions. Namely, we show that

$\text{2}\text{α+β}\text{∑}\text{t=1}\text{N}\text{c}{}_{\mathrm{i}}\text{= 1 ?}\text{1}\text{α+β}\text{∑}\text{t=1}\text{N}\text{|a}{}_{\mathrm{i}}\text{?b}{}_{\mathrm{i}}\text{|}$

, where

$\text{α =}\text{∑}\text{t=1}\text{N}\text{a}{}_{\mathrm{i}}\text{, β =}\text{∑}\text{t=1}\text{N}\text{b}{}_{\mathrm{i}}$

, and the a's, b's c's are as defined above.     

Energetics of coupled Na+ and Cl− entry into epithelial cells of bullfrog small intestine

Na⁺, K⁺ and Cl^? concentrations ($\text{c}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$) and activities ($\text{a}{}_{\mathrm{j}}{}^{\mathrm{<mtext>i</mtext>}}$), and mucosal membrane potentials ($\text{E}{}_{\mathrm{<mtext>m</mtext>}}$) were measured in epithelial cells of isolated bullfrog (Rana catesbeiana) small intestine. Segments of intestine were stripped of their external muscle layers, and bathed (at 25°C and pH 7.2) in oxygenated Ringer solutions containing 105 mM Na⁺ and Cl^? and 5.4 mM K⁺. Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry and Cl^? concentrations by conductometric titration following extraction of the dried tissue with 0.1 M HNO₃. ¹⁴C-labelled inulin was used to determine extracellular volume. $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ was measured with conventional open tip microelectrodes, $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with solid-state Cl^?-selective silver microelectrodes and $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ with Na⁺- and K⁺-selective liquid ion-exchanger microelectrodes. The average $\text{E}{}_{\mathrm{<mtext>m</mtext>}}$ recorded was ?34 mV. $\text{c}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{c}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{c}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 51, 105 and 52 mM. The corresponding values for $\text{a}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$, $\text{a}{}_{\mathrm{<mtext>K</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ and $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ were 18, 80 and 33 mM. These results suggest that a large fraction of the cytoplasmic Na⁺ is ‘bound’ or sequestered in an osmotically inactive form, that all, or virtually all the cytoplasmic K⁺ behaves as if in free solution, and that there is probably some binding of cytoplasmic Cl^?. $\text{a}{}_{\mathrm{<mtext>Cl</mtext>}}{}^{\mathrm{<mtext>i</mtext>}}$ significantly exceeds the level corresponding to electrochemical equilibrium across the mucosal and baso-lateral cell membranes. Earlier studies showed that coupled mucosal entry of Na⁺ and Cl^? is implicated in intracellular Cl^? accumulation in this tissue. This study permitted estimation of the steady-state transapical Na⁺ and Cl^? electrochemical potential differences (Δμ&#x0304;Na and Δμ&#x0304;Cl). Δμ&#x0304;Na (?7000 J · mol^?1; cell minus mucosal medium) was energetically more than sufficient to account for Δμ&#x0304;Cl (1000–2000 J · mol^?1).     

Characterization of a monoclinic crystal form of an enzyme of steroid metabolism, Δ5-3-ketosteroid isomerase

A monoclinic crystal form ($\text{P2}{}_1\text{, a = 140.4}\text{A}\text{?}\text{, b = 85.0}\text{A}\text{?}\text{, c = 94.5}\text{A}\text{?}$, β= 130.1 °) of Δ⁵-3-ketosteroid isomerase from Pseudomonas testosteroni (EC 5.3.3.1), grown at pH 7.0, has been characterized. Crystal-density measurements show that the asymmetric unit contains 12 protomers (M_r = 13,394).     

Crystallographic studies of immunoglobulins: crystallization of the Fc fragment of rabbit IgG with and without cleavage of the inter-chain disulphide bridge

The Fc fragment was prepared from rabbit immunoglobulin G by digestion with papain, both with the inter-chain disulphide bond intact, and after reduction and alkylation. These two types of Fc crystallized in different, yet related forms, each with one dimer in the asymmetric unit. The covalently linked dimer crystallized in space group $\text{P2}{}_1\text{; a = 68.85 ± 0.05}\text{A}\text{?}\text{, b = 72.50 ± 0.05}\text{A}\text{?}\text{, c = 60.40 ± 0.05}\text{A}\text{?}$ and β = 105.1 ± 0.2 °. The reduced, non-covalently linked dimer also crystallized in space group $\text{P2}{}_1\text{; a = 81.55 ± 0.05}\text{A}\text{?}\text{, b = 55.65 ± 0.05}\text{A}\text{?}\text{, c = 68.85 ± 0.05}\text{A}\text{?}$ and β = 1051 ± 0.2 °. A non-crystallographic 2-fold axis relating the two identical polypeptide chains is clearly visible in the h0l projection of the second crystal form.     

Yeast transfer RNAasp: a new high-resolution x-ray diffracting crystal form of a transfer RNA.

Systematic crystallization studies on brewer's yeast aspartic acid transfer RNA have yielded different crystal forms, one of them diffracting to 3 Å resolution. The high resolution crystal form is orthorhombic ($\text{C222}{}_1\text{, a = 61}\text{A}\text{?}\text{, b = 68}\text{A}\text{?}\text{, c = 148}\text{A}\text{?}$ with one molecule per asymmetric unit) and is stable for over four days under X-rays.     

Characterization of two new crystal forms of uteroglobin.

Two new crystal forms of oxidized uteroglobin have been obtained. An orthorhombic one ($\text{P2}{}_1\text{2}{}_1\text{2, Z = 2, a = 44.48 (5)}\text{A}\text{?}\text{, b = 36.93 (5)}\text{A}\text{?}\text{, c = 32·34 (5)}\text{A}\text{?}$) and a monoclinic one ($\text{P2}{}_1\text{, Z = 2, a = 44.56 (5)}\text{A}\text{?}\text{, b = 46.06 (5)}\text{A}\text{?}\text{, c = 37.43 (4)}\text{A}\text{?}\text{, β = 120.92 ° (5)}$). Both were grown at pH ~7.0 and diffract to a resolution of 2·1 to 2·2 Å. Data collections for native crystals have been recorded with an automatic four-circle diffractometer.     

Preliminary X-ray diffraction studies on a ferredoxin from the thermophilic archaebacterium,Thermoplasma acidophilum

A ferredoxin from the thermophilic archaebacterium, Thermoplasmaacidophilum, is supposed to contain two (4Fe-4S) active centers; one center could be linked by four cysteine residues to the protein and the other bonded with three cysteines and an unknown group. This ferredoxin has been crystallized by salting-out against 2.3 m-ammonium sulfate solution. The space group is P2₁2₁2 with cell dimensions of $\text{a = 59.20}\text{A}\text{?}\text{, b = 52.77}\text{A}\text{?}\text{and}\text{c = 41.28}\text{A}\text{?}$. Four molecules pack in the unit cell with $\text{V}{}_{\mathrm{<mtext>m</mtext>}}\text{= 2.03}\text{A}\text{?}{}^3\text{/dalton}$.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

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.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Damping of oscillations in the semiquinone absorption in reaction centers after successive flashes. Determination of the equilibrium between QA−QB and QAQB−

A quantitative model for the damping of oscillations of the semiquinone absorption after successive light flashes is presented. It is based on the equilibrium between the states Q_A^?Q_B and Q_AQ_B^?. A fit of the model to the experimental results obtained for reaction centers from Rhodopseudomonas sphaeroides gave a value of $\text{α =}\text{[}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{]}\text{([}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}{}^{\mathrm{?}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}\text{] + [}\text{Q}{}_{\mathrm{<mtext>A</mtext>}}\text{Q}{}_{\mathrm{<mtext>B</mtext>}}{}^{\mathrm{?}}\text{])}\text{= 0.065 ± 0.005}$ (T = 21°C, pH 8).