Conformation of 1- and 3-deazaadenosines in solution as studied by 1H nuclear magnetic resonance spectroscopy.

¹H nuclear magnetic resonance spectra of 1 - (II) and 3-deazaadenosines (III) together with adenosine (I) in dimethylsulfoxide have been examined. Features of coupling constants indicate that the furanose rings of I, II, and III have similar conformational preferences and that conformations about the 4′-C–5′-C bond are preferentially $\text{gauche-gauche}$. Nuclear Overhauser effect and spin-lattice relaxation-time measurements demonstrate that II predominantly adopts the $\text{syn}$-conformation similar to that of I, whereas that of III has a greater $\text{anti}$ (freely rotating) component. The results suggest that the $\text{syn}$-conformation in II as well as I is stabilized presumably through a hydrogen bond between the 3-N and 5′-hydroxyl group.     

Peroxidase activity in human red cell: a biological model for excited state molecules generation.

The presence of enzymically generated triplet acetone in red cells and energy transfer to eosin, rose bengal and 9,10-dibromoanthracene-2-sulfonate was indicate by: (1) product distribution; (2) K_ET τ_o, similar to the 2-methylpropanal/peroxidase/O₂ system; (3) correlation between hemolysis, oxygen uptake and photon emission; (4) membrane protection by energy acceptors, and (5) by comparison of the 2-methylpropanal/peroxidase/O₂ system with 2-methylpropanal/red cells/membranes/O₂ and 2-methylpropanal/acid extractable protein from red cells membrane/O₂ systems, which have a high peroxidase activity.This is the first report of a biological system producing a photohemolysis effect in the dark.     

A high-resolution c.p.-m.a.s. 13C-n.m.r. study of solid-state cyclomaltohexaose inclusion-complexes: Chemical shifts and structure of the host cyclomaltohexaose

High-resolution, solid-state ¹³C-n.m.r. spectra were obtained for several crystalline cyclomaltohexaose inclusion-complexes. The resonances of C-1, C-4, and C-6 of the host were dispersed. The averaged ¹³C shifts of these resonances were in good agreement with the ¹³C shifts observed in solution, where the dispersion due to conformational diversity is expected to be averaged by rapid interconversion of the conformers. This result indicates that the most plausible source of the solid-state ¹³C-shift dispersions of the resonances of C-1 and C-4 is the diversity of conformations about the glycosidic linkage. The molecular origins of conformation-dependent ¹³C shifts are discussed.     

Structural studies of extracellular glucans of Streptococcus mutans by proton magnetic resonance

Proton magnetic resonance spectra at 100 MHz were obtained for water-soluble and water-insoluble glucans from 11 strains of Streptococcus mutans. The percentages of α-D-(1→6) and non-α-D-(1→6)-, namely, α-D-(1→3)-, linkages were calculated from the anomeric-proton resonances in the 4.7-4.8 and 5.0-5.1 p.p.m. range, respectively. The average content of α-D(1→6) linkages in the polymer fractions precipitating from solution during synthesis of the glucans was generally much lower than that of fractions remaining in solution. The frequent appearance of the α-D-(1→3) resonances as doublets in the spectra suggested neighboring-group effects among the possible α-D-(1→3) and α-D-(1→6) linkage-configurations. These effects were confirmed from 100-MHz spectra of products of a dextranase-degraded, water-insoluble glucan, and a 270-MHz spectrum of an undegraded glucan. It was thus possible to assign the doublet resonances to α-D-(1→3), homogeneous, heterogeneous, and branch configurations, although complete differentiation among proportions of each configuration in the glucan chains could not be achieved.     

N.m.r. studies of cycloamylose inclusion-complexes with p-substituted phenols

Aqueous solutions of inclusion complexes of host cyclomaltohexa- and cyclomaltohepta-ose (cyclohexa- and cyclohepta-amylose) with guest phenols p-substituted with Br, Cl, CN, NO₂, CH₃, CHO, and COOH have been studied by ¹H- and ¹³C-n.m.r. spectroscopy. The stability of the complexes depends on the guests, the cycloamyloses, and the ionisation states of the host hydroxyl groups. The ¹H data for the host protons in the complexes show that the guests are more deeply inserted into the cavity of cyclomaltoheptaose than into that of cyclomaltohexaose.     

Interaction of short chain and long chain fatty acid phosphoglycerides and bile salts with prothrombin

An equimolar mixture of phosphatidylserine and (dioleoyl)phosphatidylethanolamine could substitute for brain cephalin preparations in the single stage prothrombin assay. However, no clot promoting activity was observed on the addition of any of the individual long chain fatty acid-containing phospholipids. Short chain fatty acid-containing phospholipids, such as diheptanoylphosphatidylcholine, diheptanoylphosphatidylethanolamine, diheptanoylphosphatidic acid, and dihexanoylphosphatidylcholine, or dihexanoylphosphatidylethanolamine were inhibitory under all conditions studied. Similar effects of these two general classes of phospholipids were observed in a two-stage thrombin generation system, in which a mixture of bovine Factor Xa, Factor Va, and Ca²⁺ were interacted with prothrombin.In the presence of 25 mM Ca²⁺, dioleoylphosphatidic acid or brain phosphatidylserine alone, and with other long chain phospholipids, formed complexes with bovine plasma prothrombin. On the other hand, dioleoyl-, diheptanoyl- or dihexanoylphosphatidylcholine under comparable conditions showed no binding to prothrombin. There appeared to be a small degree of binding of diheptanoylphosphatidic acid to prothrombin, but it was insufficient to cause any significant change in apparent molecular weight of prothrombin. A mixture of prothrombin, Factor V, diheptanoylphosphatidic acid/diheptanoylphosphatidylcholine and Ca²⁺ eluted in the void volume of Sephadex G-200, but showed a much reduced coagulant activity. Though a net negative charge on the phospholipid surface is required for phospholipid-protein interactions, this does not necessarily promote coagulant activity.Bile acids and bile salts, such as cholic acid, deoxycholic acid, taurocholic acid, glycocholic acid, lithocholic acid and dehydrocholic acid, exerted varying levels of stimulation on the prothrombin assay and thrombin generation system, but were not as effective as the phospholipids. Interestingly, no interaction of these bile acids or salts with prothrombin was noted in the presence of Ca²⁺. The results of these experiments suggest that negatively charged micelles per se are not sufficient for binding alone and that other chemical and physical characteristics of phospholipids are of prime importance.     

Overestimates of the size of poly(A) segments.

Poly(A) molecules containing on average 25, 45 and 90 nucleotide residues are all eluted from DEAE-Sephadex in the presence of 7 M urea by approximately the same NaCl concentration which is higher than that required to elute 4 S and 5 S RNA. The same poly(A) molecules have electrophoretic mobilities on 12% polyacrylamide gels which are proportional to the logarithm of the number of nucleotide residues they contain but not to the number found in 4 S and 5 S RNA, even after denaturation of the RNA and performing electrophoresis in the presence of 2.2 M formaldehyde. As a result, many reported estimates of poly(A) size derived from such techniques are probably too large and need re-evaluation. Corrections are suggested for the use of 4 S and 5 S RNA as molecular weight markers for electrophoresis on 12% polyacrylamide gels.     

Bleomycin-A2 complexes with poly(dA--dT): a proton nuclear magnetic resonance study of the nonexchangeable hydrogens

The mRNA coding for uteroglobin, a progesterone-induced uterine protein, has been partially purified from 4-day pregnant rabbit uterus. Double-stranded DNA synthesized from the partially purified mRNA preparation was inserted into the Pst I site of pBR 322. Bacterial transformants containing uteroglobin DNA sequences were identified by their ability to enrich for uteroglobin mRNA on hybridization with total uterine poly A-RNA. The identity of one recombinant was confirmed unambiguously by matching its nucleotide sequence with the amino acid sequence of the uteroglobin polypeptide.     

The interaction of Ca2+ with human factor IX

The binding isotherms of Ca²⁺ and Sr²⁺ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca²⁺, a total of 16.0 ± 1.0 sites, of K_D 7.3 ± 0.2 × 10^?4m, are present on human Factor IX. Similar analysis of the Sr²⁺ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a K_D of 1.9 ± 0.1 × 10^?3m. Both Sr²⁺ and Mn²⁺ effectively displace Ca²⁺ from human Factor IX; whereas Mg²⁺ is considerably less potent in this regard. Conversely, Ca²⁺ is capable of nearly complete displacement of Sr²⁺ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca²⁺ concentration. Sr²⁺ can substitute for Ca²⁺ in this activation process. Mn²⁺ cannot, in itself, substitute for Ca²⁺ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca²⁺. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca²⁺, Sr²⁺, and Mn²⁺ all allow activation to proceed equally well.     

The basis for EDTA-stimulation of methemoglobin reduction in hemolysates of human erythrocytes.

We have studied the stimulation by EDTA of methemoglobin reduction in hemolysates of human erythrocytes. The EDTA effect has been shown not to be the result of an allosteric interaction of EDTA with hemoglobin or the result of a photochemical reduction. The effect does not appear to be due to a direct interaction of free EDTA with either of the catalytic components of the erythrocyte methemoglobin reduction system. The EDTA stimulation seen in hemolysates is due to the formation of an iron-EDTA complex, which transfers electrons from the reductase to methemoglobin.     

A new method of acetonation. Synthesis of 4,6-O-isopropylidene-D-glucopyranose

Ethyl isopropenyl ether reacts with D-glucose in N,N-dimethylformamide containing a trace of p-toluenesulfonic acid to give crystalline 4,6-O-isopropylidene-α,β-D-glucopyranose (2) in near-quantitative yield. The structure of 2 was established by n.m.r. spectroscopy of it and of its β-triacetate 3, and by conversion of 3 through deacetonation and subsequent acetylation into β-D-glucopyranose pentaacetate (5). The acetonation reagent operates under kinetic control, with favored attack at primary hydroxyl groups, instead of by the thermodynamic control associated with conventional acetonation methods. The reagents converts methyl α-D-glucopyranoside (7) into the 4,6-isopropylidene acetal 8, and D-mannitol (9) into a 2:1 mixture of the 1,2:5,6-di-isopropylidene acetal 10 and the 1,2:3,4:5,6-tri-isopropylidene acetal 11.     

Conformational studies on aldonolactones by n.m.r. spectroscopy. Conformations of d-glucono-1,5-lactone and d-mannono-1,5-lactone in solution

The conformations of d-glucono-1,5-lactone (1) and d-mannono-1,5-lactone (2) in solution were investigated by ¹H- and ¹³C-n.m.r. spectroscopy. Conformational equilibria for 1 and 2 were found to lie strongly in favor of the ⁴H₃(d),gg and B_2,5(d),gg conformations, respectively.     

Association of nucleosidediphosphate kinase with microtubules.

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly $\text{in vivo}$ by modulating the $\text{GTP}\text{GDP}$ ratio in the microtubule environment.     

A quantitative study of organic phosphate-amine interaction by 31P nuclear magnetic resonance

Interactions between the phosphate group of 4-deoxypyridoxine 5′-phosphate and different protonated amines were quantitatively measured by means of {³¹P}-¹H nuclear magnetic double resonance technique combined with pD titration. An interaction of the phosphate group with added amine resulted in a measurable difference in the ³¹P chemical shift of these phosphate-containing samples with and without amine [Δδ(³¹P)]. Basic amino acids and biogenic amines had significant measurable Δδ(³¹P) values. No interactions were observed for acidic or neutral α, β and γ-amino acids.     

Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate.

The utilization of Fe²⁺-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The first procedure is used in standard disc electrophoresis and involves fixing the protein with trichloroacetic acid, staining with Fe²⁺-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate-containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe²⁺-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 μg of bovine serum albumin, and the limit of detection of this protein is 1 μg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fe²⁺-bathophenanthroline sulfonate is an excellent protein stain.     

2-epi-fortimicin B. Participation of 1-N-benzyloxycarbonylamino and 1-acetamido groups in solvolysis of 2-O-(methylsulfonyl)fortimicin B derivatives

Synthesis of 2-epi-fortimicin B has been accomplished by processes involving solvolyses of both 1-N-benzyloxycarbonyl- and 1-N-acetyl-2-O-(methylsulfonyl)fortimicins B, which occur with participation of the carbonyl oxygen atoms of the 1-N-acyl groups. The results illustrate both the greater effectiveness of acetamido groups in neighboring-group participation relative to benzyloxycarbonylamino groups, and the sensitivity of the nature of the products to the reaction conditions.     

Development and validation of a sensitive radioimmunoassay for naturally occurring beta-endorphin-like peptides in human plasma

β-Endorphin-like peptides in blood plasma of normal human subjects were studied by means of a radioimmunoassay (RIA) and gel filtration. Plasma was extracted with silica gel, which was washed with water and 1 n HCl, and eluted with 50% acetone. Plasma extracts thus obtained and standard synthetic human β-endorphin yielded parallel RIA curves. Total immunoreactivity in normal donors ranged from 1.2 to 10.4 fmol/ml (21 subjects). The immunoreactivity was completely destroyed by treatment with papain. Gel filtration indicated the presence of three components-one of unknown nature at the void volume and the others at elution positions characteristic of β-lipotropin and β-endorphin. Recoveries of human β-endorphin and β-lipotropin added to plasma were 53 and 58%, respectively. Addition of N-ethylmaleimide to plasma or of aprotinin to blood immmediately following collection had no effect on the amount of total immunoreactivity. Furthermore, a large amount of β-endorphin-like immunoreactivity. The above results lead us to conclude that a β-endorphin-like immunoreactive peptide occurs naturally in plasma of normal human subjects.