Oxygen equilibrium characteristics of hemoglobins of baboons, Theropithecus gelada,Papio hamadryas and Papio anubis

Hemoglobins of three baboons, Theropithecus gelada, Papio hamadryas- and Papio anubis, were purified and their oxygen equilibrium characteristics were studied. (a) Oxygen affinity, as expressed by P₅₀, oxygen partial pressure for 50% oxygen binding, was in the order of gelada hemoglobin > anubis hemoglobin > hamadryas hemoglobin although the differences were small. (b) The presence of 2,3-diphosphoglycerate reduced their oxygen affinity in a similar manner. The effect on baboon hemoglobins was greater than that on human and Japanese monkey hemoglobins. (c) The intensity of the Bohr effect, as expressed by $\text{?Δlog}\text{P}{}_{50}\text{ΔpH}$, at pH 7·4 agreed well with each other and the value was 0·62 in the presence of 2 mm diphosphoglycerate and 0·52 in its absence. These results indicate that phenotypic adaptation (acclimatory) may play an important role in the adaptation of gelada baboon to high altitudes.     

Hemoglobin Dallas (α97(G4)Asn→Lys):functional characterization of a high oxygen affinity natural mutant

Hemoglobin Dallas, an α-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1}\text{mmHg at pH}\text{7.3}\text{and}\text{20°C), diminished cooperativity (0n,}\text{the Hill coefficient}\text{= 1.7}$) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20°C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be atttributed to a more ‘relaxed’ T-state.     

Non-linear relationships between oxygen saturation and magnitude of fine structure of ultraviolet oxy versus deoxy difference spectrum in human hemoglobin

Ultraviolet difference spectra of fully oxygenated hemoglobin $\text{vs.}$ successively deoxygenated or reoxygenated hemoglobin were determined in the absence and presence of organic phosphates. Magnitude of fine structure in the difference spectrum around 290 nm, which is considered to be a partial reflection of oxygenation-induced changes in quaternary conformation of hemoglobin, was not linearly related to fractional oxygen saturation of hemoglobin of the reference cell. The non-linear feature was influenced by the organic phosphates as predicted by the allosteric model of Monod $\text{et al.}$ The present study suggests that the ultraviolet oxy $\text{vs.}$ deoxy difference spectrum measurements provide a useful way to examine the validity of the model.     

Modification of ribulose bisphosphate carboxylase by 2,3-butadione

D-ribulose-1,5-bisphosphate carboxylases purified from barley or formate-grown $\text{Pseudomonas oxalaticus}$ were inactivated by 2,3-butadione. Pseudo first-order inactivation depended on the presence of borate and was reduced by product 3-phosphoglycerate. The half-times at 30°C and pH 8.3 in the presence of 2 mM 2,3-butadione are 10 and 60 minutes for the enzymes from $\text{P. oxalaticus}$ and barley, respectively. Saturation kinetics and arginine modification were demonstrated for the enzyme from $\text{P. oxalaticus}$.     

Induction of single-strand regions in nuclear DNA by adriamycin.

Incubation of adriamycin with isolated nuclei converts nuclear DNA to a form which is susceptible to hydrolysis by $\text{Neurospora}$$\text{crassa}$ nuclease an enzyme highly specific for the cleavage of single-stranded DNA. The effect of adriamycin on nuclear DNA incubated in the presence of the nuclease can be determined by measuring the release of acid-soluble nucleotides or by analyzing the DNA after centrifugation in neutral sucrose gradients. Similar changes in chromatin structure are not observed during incubation of nuclei with adriamycin alone. In addition to adriamycin, daunomycin and ethidium bromide are also active in inducing the formation of DNA structures which are susceptible to the $\text{Neurospora}$$\text{crassa}$ nuclease. The results suggest that certain antitumor agents can induce the formation of single-strand regions in nuclear DNA and that these sites probably occur as a result of a DNA strand separating event.     

The p-cymene pathway in PseudomonasputidaPL: Isolation of a dihydrodiol accumulated by a mutant

A mutant strain (PL pT $\text{11}\text{43}$) of $\text{Pseudomonas}\text{putida}$ PL, has been isolated for its inability to growth with $\text{p}$-cymene as carbon source. The mutant oxidizes $\text{p}\text{-cymene}$ (and $\text{p}$-cumate) to a compound (λmax 293 nm) which is readily converted to 3-hydroxy-$\text{p}$-cumate by acid. 4-Trifluoromethylbenzoate is oxidized by the mutant to an acid-stable intermediate (λmax 277nm) that has been crystallized. The spectral properties (u.v., i.r., NMR and mass) of this metabolite are consistent with those expected for a 2,3-dihydro-2,3-dihydroxy derivative of 4-trifluoromethylbenzoate. Further support of this structure was provided by elemental analysis and the properties of two derivatives of the metabolite, 4-trifluoromethyl-3-hydroxybenzoate and an acetonide formed with 2,2-dimethoxypropane. The stability of a product obtained by treatment of the dihydrodiol metabolite with triacetylosmate indicates that it is the $\text{cis}\text{-isomer}$.     

An X-ray determination of the molecular interactions in hemoglobin C: a disease characterized by intraerythrocytic crystals.

The X-ray structure of cyanomet human hemoglobin C has been solved and refined, R ～27%. The molecular packing can be represented in two dimensions by two sets of parallel strands, one set in the $\text{b}$ direction and the other in the $\text{c}$ direction. Taken together the two sets of strands interconnect the molecules into square nets or layers where each molecule contacts its four nearest neighbors. Molecules in one layer are displaced in $\text{a}$ and $\text{b}$ so that they fit into the “holes” of the square arrays of the adjacent layers (normal to $\text{a}$) resulting in a pseudo body-centered cubic packing. This packing can account for the hemoglobin crystallization in and fragility of the erythrocytes. The aberrant β6A3 Lys residue is in a position to influence the crystal formation.     

Specificity of salivary-bacterial interactions: II. Evidence for a lectin on Streptococcussanguis with specificity for a NeuAcα2,3Ga1β1,3Ga1NAc sequence

Evidence is presented for the presence of a lectin on $\text{Streptococcus}\text{sanguis}$ with specificity towards the major acidic oligosaccharide of human salivary mucin. Based upon hemagglutination inhibition studies, the strongest inhibitor was NeuAcα2,3Galβ1,3GalNAcol ? NeuAcα2,3Galβ1,4Glc ? NeuAc > Gal. Interactions were not heat sensitive or charge dependent, and were not affected by the presence of bacterial cell associated neuraminidase. The lectin could be extracted from $\text{Streptococcus}\text{sanguis}$ with lithium 3,5-diiodosalicylate (LIS). Incubation of LIS extracts with carbohydrate ligands demonstrated that the specificity of binding was $\text{NeuAcα2,3Galβ1,3[}{}^3\text{H-]GalNAcol ? Galβ1,3[}{}^3\text{H-]GalNAcol}$.     

Dependence on pH of formation and oxygen affinity of hemoglobin S fibers in the presence and absence of phosphates and polyphosphates

This paper presents data on the effect of phosphates and polyphosphates on the formation of hemoglobin S fiber, and on the Bohr effect of hemoglobin S samples whose concentration was high enough (near 5 mM) in order to form fibers upon deoxygenation. The experiments were performed in 0.2 M Bistris or Tris buffers at 30 degrees C in the presence and absence of inositol hexakisphosphate and of 2,3-diphosphoglycerate. Alternatively, 0.2 M phosphate buffers were used without addition of effectors. Under these conditions, few fibers were formed in Tris or Bistris buffers, while extensive fiber formation occurred in the presence of phosphates and polyphosphates. In all cases, increasing pH strongly inhibited fiber formation. At pH 7.5 and above, fibers were not formed in our samples. In the presence of phosphates and polyphosphates fiber formation reduced the oxygen affinity of hemoglobin S with respect to either hemoglobin A or soluble hemoglobin S under similar experimental conditions. The fiber-polyphosphate complexes showed a larger Bohr effect than that in hemoglobin A. In the presence of inositol hexakisphosphate fiber-forming solutions of hemoglobin S liberated as much as six protons per tetramer upon oxygen binding. The increased liberation of protons was probably due to a higher affinity of the effectors for the fibers of hemoglobin S. Very likely the higher affinity was supported by a conformational change of hemoglobin S specific for the fibers.     

Quantitative analysis of pyridine nucleotides in red blood cells: a single-step extraction procedure.

Quantitative analysis of red cell pyridine nucleotides has been unreliable in the past because of technical problems in extracting them in the presence of hemoglobin. A simple alcoholic extraction procedure for analysis of pyridine nucleotides in red blood cells is described in this paper. Pyridine nucleotides extracted in the presence of hemoglobin in solution show recoveries of NADH, NAD, and NADP averaging over 70%, while recoveries of NADPH were about 60%. In order to show that these techniques could detect actual intracellular differences in nucleotides inside red cells, two experiments were performed in which the ratios of the nucleotides would be predictably altered. Intact cells incubated in the presence of methylene blue show a decrease in the $\text{NADPH}\text{NADP}$ ratio, and intact cells incubated in the presence of hydrazine and lactate show an increase in the $\text{NADH}\text{NAD}$ ratio. The changes in pyridine nucleotide ratios in these experiments are in the expected direction and were easily detected. Levels of pyridine nucleotides in red blood cells of normal human adults are also presented.     

Effect of specific inhibitors on mitochondrial DNA replication in HeLa cells

The crystal structure of an orthorhombic form of 2′-0-methyl cytidine was determined from three dimensional X-ray diffraction data. The two molecules in each asymmetric unit have C2-$\text{endo}$ C3-$\text{exo}$ puckered furanose rings. This differs from the C3-$\text{endo}$ puckering observed for cytidine (1) and it may have some relevance to the kinks that appear at the two 2′-0-methylated nucleotides in the anticodon phosphate ester backbone of the phe tRNA structure (2). This work and other studies (3,4) show that the presence of a 2′-0-methyl group does not prevent the furanose moiety from adopting its most commonly observed configurations. 2′-0-methyl nucleotides make up a small percentage of the residues in HnRNA, rRNA, tRNA and mRNA and therefore their conformational nuances are of interest.     

1H and 31P nuclear magnetic resonance investigation of the interaction between 2,3-diphosphoglycerate and human normal adult hemoglobin

High-resolution 1H and 31P nuclear magnetic resonance spectroscopy has been used to investigate the binding of 2,3-diphosphoglycerate to human normal adult hemoglobin and the molecular interactions involved in the allosteric effect of the 2,3-diphosphoglycerate molecule on hemoglobin. Individual hydrogen ion NMR titration curves have been obtained for 22-26 histidyl residues of hemoglobin and for each phosphate group of 2,3-diphosphoglycerate with hemoglobin in both the deoxy and carbonmonoxy forms. The results indicate that 2,3-diphosphoglycerate binds to deoxyhemoglobin at the central cavity between the two beta chains and the binding involves the beta 2-histidyl residues. Moreover, the results suggest that the binding site of 2,3-diphosphoglycerate to carbonmonoxyhemoglobin contains the same (or at least some of the same) amino acid residues responsible for binding in the deoxy form. As a result of the specific interactions with 2,3-diphosphoglycerate, the beta 2-histidyl residues make a significant contribution to the alkaline Bohr effect under these experimental conditions (up to 0.5 proton/Hb tetramer). 2,3-Diphosphoglycerate also affects the individual hydrogen ion equilibria of several histidyl residues located away from the binding site on the surface of the hemoglobin molecule, and, possibly, in the heme pockets. These results give the first experimental demonstration that long-range electrostatic and/or conformational effects of the binding could play an important role in the allosteric effect of 2,3-diphosphoglycerate on hemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Inhibitory effect of iron on the uptake of lead by erythrocytes.

It is well known that more than 90% of the lead found in blood is associated with the erythrocytes. The present $\text{in vitro}$ experiments show that the uptake of lead-203 by rabbit erythrocytes is inhibited by the presence of non-radioactive lead or iron or by reduction of the incubation temperature. The inhibitory effect of iron on radioactive lead uptake by erythrocytes is also demonstrable $\text{in vivo}$.When lead-203 is incorporated into erythrocytes $\text{in vitro}$, about 10% of the radioactivity is attached to the membrane and the remainder is found in the cytoplasm associated with hemoglobin and an unidentified low molecular weight intracellular component. In the presence of 25 μg/ml of added iron (Fe⁺⁺⁺) the uptake of radioactive lead by erythrocytes is reduced to 21.7±5.1% and membrane binding accounts for approximately 5% of this total. Chromatographic analyses of hemolysates show that the reduction in cytoplasmic labeling is directly related to decreased lead binding to the low molecular weight component, since hemoglobin binding remains unchanged.This work suggests that in addition to the interaction between iron and lead which occurs during the biosynthesis of heme, these metals may directly compete for specific erythrocyte binding sites.     

Glycerol conformation and molecular packing of membrane lipids: The crystal structure of 2,3-dilauroyl-d-glycerol

The single-crystal structure of 2,3-dilauroyl-d-glycerol has been determined by Patterson rotation and translation methods and refined to R = 0.069. 2,3-dilauroyl-d-glycerol crystallizes in the monoclinic space group P2₁, with unit cell dimensions: $\text{a = 5.46}\text{A}\text{?}\text{, b = 7.59}\text{A}\text{?}\text{, c = 34.2}\text{A}\text{?}\text{and}\text{β = 93.1 °}$, and with two molecules per unit cell. The molecules have their hydrocarbon chains aligned parallel, and are arranged in a bilayer structure. The chain stacking is achieved by a bend in the fatty acid. The hydrocarbon chains pack according to the orthorhombic perpendicular chain packing mode, and are tilted 26.5 ° from the layer normal.The structural features of 2,3-dilauroyl-d-glycerol have been analysed with reference to the corresponding hydrophobic moieties in the crystal structures of different membrane lipids. The glycerol group in 2,3-dilauroyl-d-glycerol is oriented parallel to the layer plane, but changes to an approximately layer-perpendicular orientation when a polar group is attached. The molecular conformation of the glycerol-dicarboxylic ester group, however, is identical in both the absence and presence of a head group, indicating extensive conformational restrictions for this group due to both intrinsic properties and chain stacking. The gathered data provide detailed information on the structural properties of the hydrophobic moiety of membrane lipids.     

Retinoic acid induces neuronal differentiation of a cloned human embryonal carcinoma cell line in vitro

The human embryonal carcinoma cell lines $\text{NT2}\text{D1}$ and $\text{NT2}\text{B9}$, clonally derived from Tera-2, differentiate extensively in vitro when exposed to retinoic acid. This differentiation is marked by the appearance of several morphologically distinct cell types and by changes in cell surface phenotype, particularly by the disappearance of stage-specific embryonic antigen-3 (SSEA-3), which is characteristically expressed by human EC cells. Among the differentiated cells are neurons, which form clusters interconnected by extended networks of axon bundles, and which express tetanus toxin receptors and neurofilament proteins. These observations constitute the first instance of extensive somatic differentiation of a clonal human EC cell line in vitro.     

Modulation of 2,3-diphosphoglycerate 31P-NMR resonance positions by red cell membrane shape

Na⁺ transport in the red cells of the dog is dependent on cell volume, a 20% change in cell volume leading to a 25-fold increase in apparent Na⁺ flux; the effect is dependent upon metabolic energy. We have found that swelling and shrinking dog red cells causes a shift in the ³¹P-NMR peak of 2,3-diphosphoglycerate, which is present in dog red cells at 5.5 mM. Control experiments indicate that the 2,3-diphosphoglycerate resonance peak shifts may not be attributed to: interaction with hemoglobin, changes in cell pH, ionic strength, diamagnetic susceptibility or small changes in the Mg²⁺/2,3-diphosphoglycerate ratio. Experiments with chlorpromazine and pentanol which alter red cell membrane area by a mechanism different from osmotic swelling suggest that 2,3-diphosphoglycerate interacts with a binding site in the cell that is dependent upon the physical condition of the dog red cell membrane.     

Induction by ouabain of hemoglobin synthesis in cultured friend erythroleukemic cells

Induction of erythroid differentiation in ouabain-resistant murine erythroleukemia cells by ouabain is reported. Ouabain induction results in the appearance of hemoglobin-containing cells 12–24 hr earlier than induction of the same clone by dimethyl sulfoxide. The levels of globin mRNA after ouabain induction are similar in amount to the globin mRNA levels observed after induction by dimethyl sulfoxide. The concentration of ouabain required to induce hemoglobin synthesis depends upon the K⁺ ion levels in the culture medium. Lowering the extracellular K⁺ ion concentration 2–4 fold reduced by 10–40 fold the ouabain concentration necessary for the induction of hemoglobin synthesis. In low K⁺ medium (1.8 mM), ouabain is an effective inducer of hemoglobin synthesis at a concentration of 0.02 mM. This K⁺ effect is specific for ouabain induction, since induction by other inducers, such as dimethyl sulfoxide and dimethyl acetamide, does not exhibit this marked sensitivity to the levels of K⁺ ions in the culture medium. These results suggest that the binding of ouabain to the plasma membrane enzyme, $\text{Na}\text{K}$ ATPase, is required for the induction of erythroid differentiation by ouabain. A small but significant proportion of wild-type, ouabain-sensitive cells also can be induced by ouabain, below ouabain concentrations that are toxic to these cells. The observation that the binding of ouabain to the $\text{Na}\text{K}$ ATPase induces hemoglobin synthesis suggests that changes in the intracellular concentration of K⁺ ions may be involved in the control of erythroid differentiation in Friend erythroleukemic cells.