The crystal structure of cholesteryl octanoate

Crystals of cholesteryl octanoate (C₃₅H₆₀O₂) are monoclinic, space group P2₁, with $\text{a = 12.80(3), b = 9.20(2), c = 14.12(3)}\text{A}\text{?}$, β = 93.81(3)° and 2 molecules per unit cell. The structure has been determined by Patterson rotation and translation methods from the X-ray intensities (Mo-Kα radiation) of 1320 reflections ($\text{sin}\text{θ/λ < 0.59}\text{A}\text{?}{}^{\mathrm{?1}}$) measured with a diffractometer. Refinement by block diagonal least squares and Fourier methods gave R = 0.096. The molecules are arranged in monolayers with their long axes antiparallel and severely tilted (28°). There is a close packing of cholesteryls within the monolayers, but the octanoate chains which form the layer interface regions are conformationally and thermally disordered. The crystal structure is quite different from that of cholesteryl nonanoate, as expected from the discontinuity in thermodynamic properties and phase behaviour which occurs at this point in the homologous series.     

The composition-dependence of the extent of reaction between a multivalent acceptor and a bivalent ligand: Implications of self-interaction of the ligand in precipitin effects

Theory is presented relating to the reversible interaction of an f-valent acceptor, A, with a bivalent ligand, B, which leads to the formation of a series of complexes comprising networks of alternating A and B molecules. An explicit expression is derived for the overall extent of reaction in terms of the total molar concentrations of reactants $\text{(}\text{m}{}_{\mathrm{A}}$ and $\text{m}{}_{\mathrm{B}}\text{)}$, the valency of the acceptor and the site-binding constant, k, governing the equilibria. It is shown by differentiation of this expression holding $\text{m}{}_{\mathrm{A}}$ (or $\text{m}{}_{\mathrm{B}}$) fixed that relations are available for the independent evaluation of f and k from a combination of precipitin and radioimmunoassay experiments. Moreover, it is established that dilution with solvent ($\text{m}{}_{\mathrm{A}}\text{/}\text{m}{}_{\mathrm{B}}$ fixed) cannot lead to the appearance of a precipitate with this type of crosslinking system. The latter observation forms the background for the development of theory pertaining to the joint operation of ligand dimerization, 2B?B₂, and crosslinking of the multivalent acceptor with bivalent B₂. The theoretical examination of this system is developed in terms of site-probability functions and involves the delineation of unique solutions for the extent of crosslinking reaction aided by the definition of the extent of binding in defined limits. It is shown with the use of numerical examples that the system involving self-associating ligand may result in the appearance of a precipitate on dilution with solvent and the conditions for the operation of this phenomenon are elucidated. It is noted that other types of ligand self-interaction may lead to similar effects in crosslinking systems, and the general principles emerging from this study are discussed in terms of systems in which antibody ligands are known to be involved in association reactions or are suspected to be so involved on the basis of precipitation effects observed on dilution with solvent.     

Binding equations for interacting systems comprising multivalent acceptor and bivalent ligand: application to antigen-antibody systems

Consideration is given to the reversible interaction of a bivalent ligand, B, with a multivalent acceptor, A (possessing f reactive sites) which leads to the formation of a series of complexes, A_iB_j, comprising networks of alternating acceptor and ligand molecules. A binding equation is derived on the basis of a site association constant, k, defined in terms of reacted site probability functions. This equation, which relates the binding function, r (the moles of ligand bound per mole of acceptor) to the concentration of unbound ligand, m_b, is used to show that plots of r vs. 2km_B constructed with fixed but different values of $\text{k}\text{m}{}_{\mathrm{A}}$ intersect at the point ($\text{m}{}_{\mathrm{B}}\text{=}\text{1}\text{2k}\text{, r =}\text{f}\text{2}$) where the extent of reaction and the concentrations of those complexes for which $\text{j}\text{i}\text{=}\text{f}\text{2}$ attain maximal values. Corresponding Scatchard plots are shown by numerical example to be non-linear, their second derivative being positive for all r. It follows that such deviations from linearity cannot be taken alone as evidence for site heterogeneity in cross-linking systems. The binding equation obtained directly is shown to be identical with that obtained with f = 2 by summation procedures involving the general expression for concentrations of complexes, m_AiBj, formulated in terms of appropriate statistical factors. In this way, previous findings on precipitation and gel formation in cross-linking systems are correlated with the present development of binding theory.     

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.     

Interactions between polynucleotides and platinum (II) complexes

Reaction of either $\text{cis}$ or $\text{trans}$ Pt(NH₃)₂Cl₂ with poly(A) in dilute aqueous solution leads to quantitative precipitation of the polymer at Pt/nucleotide ratios above 0.5. It is proposed that at ratios less than this, intramolecular binding of one Pt to two bases is favored; at higher ratios, intermolecular cross-linking becomes important and precipitation results. The absence of isomer selectivity in precipitation implies that the biological specificity of the $\text{cis}$ form results from a process other than cross-linking of polynucleotide strands. Other observations suggest that the coordinated ammonia of nucleotide-platinum(II) ammine complexes may be unusually labile.     

Corrections to Nomenclature of Phosphorus-Containing Compounds of Biochemical Importance

Crystals of cholesteryl oleate (C₄₅H₇₈O₂) are monoclinic, space group P2₁, with $\text{a = 12.65(3), b = 9.13(3), c = 18.79(5)}\text{A}\text{?}\text{, β = 93.3(3)°}$ and have 2 molecules in the unit cell. The crystal structure has been determined by Patterson and Fourier methods at a resolution $\text{d}{}_{\mathrm{<mtext>min</mtext>}}\text{= 1.1}\text{A}\text{?}$, using 799 X-ray intensities (CuKα) measured by a diffractometer. Structure refinement by block-diagonal least squares gave R = 0.12. The oleate chains are almost straight except for a kink at the cis-double bond. The chains pack side by side but without a regular sub-cell structure, in a manner which might be similar to the arrangement within biological membranes. As in cholesteryl octanoate, the cholesteryl ring systems pack together with extensive overlap of anti-parallel nearest neighbours. Projecting methyl groups interlock.     

Enzymes of nitrogen metabolism in legume nodules: Partial purification and properties of the aspartate aminotransferases from lupine nodules

The two aspartate aminotransferase isoenzymes, AAT-P₁ and AAT-P₂, found in the plant cytosol fraction of lupine nodules have been separated and partially purified. Both isoenzymes showed a broad pH optimum between 7.0 and 9.5 and demonstrated high substrate specificity. Molecular weights determined by gel filtration chromatography were 105,000 and 96,000 for AAT-P₁ and AAT-P₂, respectively. AAT-P₁ demonstrated a subunit molecular weight of 47,000 and AAT-P₂, 45,000. Both isoenzymes showed oxaloacetate substrate inhibition and gave similar K_m values for aspartate (2.2 and 2.6 mm) and 2-oxoglutarate (0.26 and 0.2 mm) for AAT-P₁ and AAT-P₂, respectively. However, AAT-P₂ showed a fivefold lower K_m for oxaloacetate (0.02 mm) and a 1.8-fold lower K_m for glutamate (12 mm) than did AAT-P₁ for these substrates. The parallel nature of the $\text{1}\text{V}\text{vs}\text{1}\text{[}\text{S}\text{]}$ plots together with the product inhibition kinetics were consistent with a ping-pong-bi-bi mechanism of action. The results are discussed in terms of the possible physiological significance of these plant aspartate aminotransferases in ammonia assimilation in lupine nodules.     

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.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

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.     

Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

Turnover of prolyl hydroxylase and an immunologically related protein in rabbit tissue

The turnover rates of prolyl hydroxylase and immunologically related (cross reacting) protein were examined using labeled leucine as precursor or by measuring the decay of elevated prolyl hydroxylase and immunologically cross-reacting protein back to basal levels. Prolyl hydroxylase and immunologically cross-reacting protein were purified from neonatal rabbit skin at various times following the administration of [³H]leucine. Prolyl hydroxylase was purified by affinity chromatography. Immunologically cross-reacting protein was purified by antibody precipitation from the dialyzed 70% (NH₄)SO₄ supernatants and subsequent electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide slab gels. The radioactivity of the species isolated, which corresponded to the two major subunits of prolyl hydroxylase, was used in the turnover studies of immunologically cross-reacting protein. The peak incorporation of label into prolyl hydroxylase was found to be 12 h while for immunologically cross-reacting protein this occured within 2 h. The loss of radioactivity from these protein pools denotes an apparent $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for prolyl hydroxylase of 73 h and a $\text{1}\text{2}$ for immunologically cross-reacting protein of 53 h. From the specific activity of free skin leucine pools, the effect of reutilization could be corrected and a true $\text{t}\text{1}\text{2}$ for prolyl hydroxylase of 45 h was determined. The $\text{t}\text{1}\text{2}$ values of these proteins were determined by a second method in which prolyl hydroxylase and immunologically cross-reacting protein in the aorta and liver of adult male rabbits were elevated by daily epinephrine-thyroxine treatment for 12 days. The decline of prolyl hydroxylase and immunologically cross-reacting protein with termination of treatment in the aorta denotes values of 42 h for enzyme and 53 h for immunologically cross-reacting protein. Calculated enzyme κ_d values, by both methods, indicate that breakdown of enzyme does not account for tissue immunologically cross-reacting protein.     

Preliminary X-ray studies on Pseudomonas isoamylase

Single crystals of Pseudomonas isoamylase (M_r 95,000) belong to space group P2₁2₁2₁ with unit cell dimensions of $\text{a = 137.9}\text{A}\text{?}\text{, b = 52.9}\text{A}\text{?}\text{, c = 151.2}\text{A}\text{?}$. A Guinier plot of the X-ray small-angle scattering of the protein solution gave the radius of gyration of the molecule as 27.5 Å.     

Crystallography of Bacillus subtilis levansucrase

Levansucrase, an exocellular enzyme, has been isolated from a high producer mutant, the BS5C4 constitutive strain, of Bacillus subtilis. Three crystalline forms have been obtained, all three belonging to the orthorhombic space group P2₁2₁2₁. The most suitable form for a three-dimensional structure investigation has cell dimensions, $\text{a = 68}\text{A}\text{?}$, $\text{b = 125}\text{A}\text{?}$, $\text{c = 54}\text{A}\text{?}$. There is one molecule in the asymmetric unit.     

Correlation of the perceived texture of random coil polysaccharide solutions with objective parameters

A wide range of concentrated random coil polysaccharide solutions have been assessed for textural attributes by a trained sensory panel. The only textural terms invoked to describe these model systems were ‘thickness’ and ‘stickiness’, which were shown to be highly correlated, and essentially identical numerically, using a ratio scaling technique. Viscosity (η) measurements over a wide range of shear rates (γ) for all these samples gave flow curves (log η versus log γ) of the same form. Differences in flow behaviour between samples could then be characterised completely by two parameters, the maximum viscosity at low shear rates (η₀), and the shear rate ($\text{γ}\text{?}{}_{\mathrm{0·1}}$) at which $\text{η =}\text{solη}{}_0\text{10}$. A simple linear relationship was demonstrated between these two parameters and perceived thickness (T) or stickiness (S), irrespective of polysaccharide type. For Newtonian liquids, log T (or log S) varied linearly with log η. Hence the effective ‘in-mouth’ thickness of random coil polysaccharide solutions, in normal viscosity units, may be predicted directly from η₀ and $\text{γ}\text{?}{}_{\mathrm{0·1}}$ by the simple relationship: $\text{log}\text{η}{}_{\mathrm{N}}\text{= 1·13}\text{log}\text{η}{}_0\text{+ 0·45}\text{log}\text{γ}\text{?}{}_{\mathrm{0·1}}\text{? 1·72}$ where η_N is the viscosity of a Newtonian solution which would be perceived as identical in thickness (and stickiness) to the polysaccharide solution.     

Estimation of mutation rates in cultured mammalian cells

The factors that affect reliable estimations of mutation rates (μ) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (N_t) and the number of parallel cultures (C) on the variation in the rate estimates ($\text{μ}$) inferred from the P₀ method. The analysis can be made after the derivation of the variance of $\text{μ}$, which is a measure of variation of $\text{μ}$ for a given combination of N_t and C in a number of repeat experiments. The variance of $\text{μ}$ is inversely proportional to C and to the square of N_t. N_t determines the probability of occurrence of mutation in a cell culture. By influencing the size of P₀, N_t also determines whether a rate estimate is obtainable from the experiment. Since P_o is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of $\text{μ}$. The rate estimated from $\text{P}\text{?}{}_0$ is biased, but the bias is in general 2 orders of magnitude smaller than $\text{μ}$. By the selection of an appropriate combination of N_t and C for the experiment, this bias can be reduced even further.Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis.     

Foundations of the use of an enzyme-kinetic analogy in cell-mediated cytotoxicity

A mathematical model of the ⁵¹Cr-release microcytotoxicity assay is utilized to find conditions under which the kinetics of this assay resemble the kinetics of a classical enzyme-substrate reaction. Assuming a steady-state approximation, that “bystander” effector cells do not bind markedly better than the cytotoxic effector cells, and that the programming of the target cells for lysis is irreversible, it is shown that the velocity of label release is v = v_maxT_T/(K_{$\text{1}\text{2}$}+T_T), where both V_max and K_{$\text{1}\text{2}$} are linear functions of the effector-cell population and T_T is the initial target-cell population. Moreover, the expressions for K_{$\text{1}\text{2}$} and V_max are expressed in terms of natural kinetic parameters of the process and attributes of the noncytotoxic bystanders.     

Preliminary crystallographic data for lysozyme produced by Streptomyces erythraeus.

Crystals of lysozyme from Streptomyces erythraeus are orthorhombic, space group P2₁2₁2₁ with unit cell dimensions: $\text{a = 50.01}\text{A}\text{?}\text{, b = 61.98}\text{A}\text{?}$ and $\text{c = 67.85}\text{A}\text{?}$; and one molecule, of molecular weight about 18,500, per asymmetric unit.     

The structures of barium hexafluorosilicate and cesium hexafluororhenate(V)

The crystal structure of CsReF₆, together with a reinvestigation of that of BaSiF₆, is reported. Both have been determined from single crystal three-dimensional X-ray diffraction data. The structure of BaSiF₆ has been found to conform to the initially assigned space group R$\text{3}$m, contrary to the suggestions of other workers. The unit cell of BaSiF₆ has the dimensiona a_hex 7.189(1), c_hex 7.015(1) Å; Z = 3. Refinement by a least squares method gave R 0.0079 and R_w 0.0077. Crystals of CsReF₆ belong to the lower symmetry rhombohedral space group R$\text{3}$. The unit cell has the dimensions a_hex 7.853(1), c_hex 8.140(1) Å; Z = 3. Refinement gave R 0.031 and R_w 0.030. The lowering of symmetry is caused by rotation of the ReF₆^? octahedra about the 3-fold axis through each Re atom, causing CsReF₆ to have the KOsF₆ structure.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.