Crystallisation of tRNALeuCUG from Escherichia coli after purification with hydroxyapatite.

tRNA_CUG^Leu from E. Coli was purified by column chromatography on benzoylated DEAE-cellulose, followed by hydroxyapatite prepared by an improved method. Crystals obtained by vapour diffusion gave X-ray diffraction out to 7 Å in the hk0 projection and 10 Å in h0?. The space group was P4₂2₁2 with $\text{a = b = 133}\text{A}\text{?}$, $\text{c = 66}\text{A}\text{?}$ and 8 molecules in the unit cell. Birefringence showed preferred orientation of RNA helical regions in the ab plane.     

Hyaluronic acid: a double-helical structure in the presence of potassium at low pH and found also with the cations ammonium, rubidium and caesium.

An anti-parallel double-helical structure is proposed for hyaluronic acid in the presence of the cations K ⁺, NH⁺₄, Rb⁺ and Cs⁺. In particular the crystalline phase containing potassium ions, and in a defined pH range, has been determined and refined against the X-ray diffraction amplitudes. The reflections in the diffraction pattern index on a tetragonal unit cell (a = b = 1.714 nm, c (fibre axis) = 3.28 nm) with observed systematic absences of the form h + k + l = 2n for general hkl reflections and l = 4n (where n is an integer) for 001 reflections. Two double-helical molecules pass through the unit cell at positions ($\text{1}\text{4}\text{,}\text{1}\text{4}\text{, 0}$) and ($\text{3}\text{4}\text{,}\text{3}\text{4}\text{, 0}$) as defined by space group I4₁22. Each chain forms a contracted (axial advance per disaccharide repeat of 0.82 nm) 4-fold left-handed helix which intertwines with a neighbouring chain of opposite polarity about a common axis. Features of the structure are the positioning of the carboxyl group toward the core of the double helix and the presence of hydrophobic and hydrophilic pockets between adjacent double helices. The state of protonation of the hyaluronate molecule is discussed in the light of the infrared evidence. Common physicochemical features of these four cations which promote this particular doublehelical conformation for hyaluronate are considered.     

Electron exchange coupling in a naturally occurring tetramangano cluster in the mineral helvite, (Mn4S)(SiBeO4)3

The mineral helvite, (Mn₄S)(BeSiO₄)₃, contains discrete tetrahedral Mn₄S⁺⁶ clusters in which the S^?2 is tetrahedrally coordinated and each Mn(II) is in a distorted tetrahedron of one S^?2 and three oxygens; the cluster is situated within an encompassing lattice of SiO₄^?4 and BeO₄^?6 tetrahedra. Mn₄S⁺⁶ centers provide an interesting model for comparison to the polynuclear manganese center that is associated with photosynthetic water oxidation. Magnetic susceptibility data between 77 and 298 K have been measured for a natural helvite sample containing principally Mn₄S⁺⁶ centers but with significant contamination from Mn₃FeS⁺⁶ and Mn₃CaS⁺⁶. The data exhibited Curie-Weiss behavior with μ_eff = 5.969 B.M. and θ = 178.3 K. An analysis of the magnetic susceptibility, based on Van Vleck's formalism, demonstrated the presence of antiferromagnetic coupling, with a coupling constant J = ?5.83 cm^?1. Mössbauer spectra of Mn₃FeS centers in helvite and of Fe₄S centers in the related mineral danalite have also been recorded. Isomer shifts show little temperature dependence and lie in the range 1.23–1.43 $\text{mm}\text{sec.}$. This range is typical of tetrahedrally coordinated Fe(II) in several ionic crystals but is significantly above that of Fe(II) in ferredoxins and below that in the [quinone-Fe(II)-quinone] complex of the photosynthetic bacterium,Rhodopseudomonas sphaeroides. Quadrupole splittings are highly temperature dependent, ranging from 2.4 $\text{mm}\text{sec}$ at 4.2 K to less than 0.5 $\text{mm}\text{sec}$ at 248 K.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Covalent binding of proteins and glucose-6-phosphate dehydrogenase to cellulosic carriers activated with s-triazine trichloride

A method was developed for covalently binding proteins and enzymes to cellulosic carriers such that the enzymes retained high specific activity. Optimal conditions for activating the carriers with s-triazine trichloride were found to be: (a) pretreatment of cellulose with 3 m NaOH; and (b) reaction with 5% ($\text{w}\text{w}$) s-triazine trichloride in dioxane-xylene (1:1 $\text{w}\text{w}$) for 30 min at room temperature. All proteins tested bound most readily at pH values below pH 7. Extensive investigation of immobilized glucose-6-phosphate dehydrogenase showed that: (a) over 80% of the specific activity of the enzyme was retained; and (b) the pH optimum and K_m values were not altered significantly from that of the free enzyme. The binding method has been applied successfully to hexokinase, phosphorylase and pronase.     

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively filled by a reversible consecutive dissociation-unfolding mechanism according to Na = 4M ? 4M* (where N is the native letramer, and M and M* two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101). At P ? 1 kbar, the pressure deactivalion of both isoenzymes (H₄ and M₄) is described by the two-state equilibrium N ? 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M₄: Δg_Diss = 110 $\text{kJ}\text{mol}$, ΔSDiss = ?860 J/K per mol, ΔH_Diss = ?124 $\text{kJ}\text{mol}$ (enzyme concentration 10 $\text{μg}\text{ml}$, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be ΔV_Diss = ?420 $\text{ml}\text{mol}$ (0.7 < p < 0.9 kbar). Measurements using 8-anilino-1-naphlhalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurizarion beyond 1 kbar. Within the range of reversible deactivation (p < 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. ²H₂O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

An analysis of the change in K-permeability on depolarization in terms of affinity and numbers of total channels.

A theoretical relation between permeability and ionic concentrations in a bathing solution has been derived by assuming that only channels unoccupied by a competing non-permeable ion can transport ions specific for that channel. The affinities of the channel to the ion and the competitor are expressed by dissociation constants of the ion-site and competitor-site complexes in the channel.Analyses of the relation of K permeability to [K]_o obtained from myelinated nerve fibres and Nitella cells revealed that the affinity of sites in K channels was independent of membrane potential, whereas K conductivity increased with depolarization. The value of the dissociation constant of the K⁺-site complex, K₁, was estimated as 1244 mm for myelinated nerve, and $\text{K}{}_1\text{exp(ψ}{}_0\text{F}\text{RT}$) for Nitella was 17.5 mm (ψ₀ is the surface potential at the outer surface of membrane). The dependence on voltage of the total number of K channels was estimated from the dependence of K conductance on membrane potential at [K]_o = [K]₁ (obtained from the theoretical magnitude of K current computed by using the dissociation constants described above). It should be noted that when the channels are partially saturated with K⁺, neither the chord conductance nor the “permeability coefficient”, as defined in the Goldman and Hodgkin-Katz formulation, correctly represents the dependence on membrane potential of the total number of channels.     

The enzyme "aldehyde oxidase" is an iminium oxidase. Reaction with nicotine delta 1'(5') iminium ion.

The second of the two reaction steps involved in the metabolic transformation of (?)-nicotine to (?)-cotinine $\text{(}\text{3}\text{) (}\text{i.}\text{e.}$, the oxidation of the intermediate $\text{2}\text{)}$ is mediated mainly, if not solely, by the enzyme aldehyde oxidase (EC 1.2.3.1). Of the molecular species that constitute $\text{2}$, nicotine Δ^1′(5′) iminium ion $\text{(}\text{2a}\text{)}$ appears to serve as the substrate. The enzyme has a strong affinity for $\text{2a}$, as shown in a study on the inhibition of the oxidation of 3-(aminocarbonyl)-1-methylpyridinium chloride. This study gave a value of K_i = 6 μM; K_m = 2 μM (pH 7.4). Mainly in view of this finding, “iminium oxidase” seems to be a more adequate name than “aldehyde oxidase” for this enzyme.     

Crystals of a bovine neurophysin II-dipeptide amide complex.

Bovine neurophysin II, a hormone carrier protein, has been crystallized as a binary complex with l-phenylalanyl-l-tyrosine amide, a peptide known to bind neurophysin at its hormone binding site. The crystals belong to space group P2₁2₁2₁ with cell constants of $\text{a = 121.6(10)}\text{A}\text{?}\text{, b = 67·9(6)}\text{A}\text{?}\text{and}\text{c = 62·1(6)}\text{A}\text{?}$. The density of the crystal is 1.156 g/cm³. There is a tetramer or two dimers in an asymmetric unit.     

Preliminary crystallographic data for cross-linked (lysine7-lysine41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N^ε,N^ε′-(2,4-dinitrophenylene-1,5)-(lysine⁷-lysine⁴¹)-RNase A, has been crystallized by dialysis against 30% ($\text{v}\text{v}$) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2₁2₁2₁, $\text{a = 37.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}$, with one molecule in the Crystallographic asymmetric unit.     

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S₁ and S₂ states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S₂ state. Low-temperature illumination of samples prepared in the S₂ state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH₂OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl^? with F^? exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a $\text{S =}\text{5}\text{2}$ spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

Linear oligopeptides. 56. Critical size for development of β-and α-helical structures of monodispersed homo-l-oligomethionines in the solid state

The infrared absorption properties of monodispersed, chemically and optically pure ‘amino-PEG’-bound linear homooligopeptides having the general formula $\text{t-}\text{Bx(}\text{l}\text{-Met)}{}_{\mathrm{n}}\text{NHPEG}$, n = 1–15 and H₂⁺(l-Met)_nNHPEG, n = 1–14 have been investigated in the solid state. The critical sizes for development of the β-structure and α-helix (n = 3 and n = 13, respectively), and the effect, on the latter, of the solvent from which the solid samples are cast, have been established.     

Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, v_j, within the lipid matrix. At T = 35°C a value of v_j = 1.6 · 10³ s^?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 10⁸ s^?1 and 1.5 · 10⁸ s^?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells ($\text{τ}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{min at}\text{T = 37°}\text{C}$) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 100}\text{min at}\text{T = 37°}\text{C}$.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.