The relationship between the osmotic fragility of human erythrocytes and cell age

Erythrocytes in a normal blood sample are hemolyzed over a range of hypotonic salt concentrations. In order to investigate the relationship between the distribution of osmotic fragilities and the distribution of cellular ages, the osmotic fragility has been compared with three indices of cellular age. The activity of glutamic-oxalacetic transaminase (GOT) and the percentage hemoglobin A_1C were measured in samples hemolyzed in different hypotonic salt concentrations. The osmotic fragility curve was also obtained for cells of different density separated by centrifugation. These experiments indicate that the fragility distribution is not an accurate reflection of the distribution of cellular ages. The mean fragility for older cells is higher than that of younger cells. However, cellular aging does not produce a gradual increase in osmotic fragility. Instead, it seems to produce changes which can both increase and decrease the fragility, resulting in a broader distribution of fragilities with some of the older cells actually less fragile than the younger ones.     

Glutathione-related inhibition of prostaglandin metabolism

Placental homogenates contain a heat-stable, dialyzable fraction which specifically inhibits two placental enzymes, both of which possess 15-hydroxyprostaglandin dehydrogenase and 9-ketoprostaglandin reductase activities. The inhibition of the two enzymes is the same. The inhibitor has been resolved into two components by gel filtration on a column of Sephadex LH-20. The component which eluted first has been identified as oxidized glutathione (GSSG), the other as a glutathione-containing material (GSX). Inhibition of the 15-hydroxyprostaglandin dehydrogenase activity is competitive with respect to the prostaglandin substrate (K_i^GSSG = 26 μM, K_i^GSX = 1.4 μM). Inhibition of the 9-ketoprostaglandin reductase activity is also competitive with respect to the prostaglandin substrate (K_i^GSSG = 68 μM). The most effective inhibitor of the 15-hydroxyprostaglandin dehydrogenase is the prostaglandin A₁-glutathione adduct (K_i = 0.27 μM). This compound is not a substrate for oxidation of the 15-hydroxyl group but it is the best substrate found to date for reduction of the 9-keto function.     

Ornithine metabolism in relation to stimulation of urea cycle,induced by high protein diet

Approximate rates of some in vivo ornithine metabolisms in rats were calculated by pulse-labeling data, on the assumption that hepatic metabolite levels are constant during a given 3-min period. The rate of ornithine catabolism was 70–110 nmol/min/g liver; that of urea output was 3–6 μmol/min/g liver; the rotary rate of the “ornithine flux” was 10–12 rpm. A change from a 25 to a 70% casein diet resulted in a 1.5-fold augmentation in the rate of ornithine catabolism and a 1.6-fold increase in the rate of urea output; however, the rate of the “ornithine flux” remained nearly constant These findings suggest that stimulation of the urea cycle is accompanied not by acceleration of the cycle rotation, but rather by increased mass of the metabolite flux.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

Studies on the characterization of human erythrocyte acetylcholinesterase and its interaction with antibodies

Human erythrocyte membranes were solubilized in 5% Triton X-100 and the acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was isolated by affinity chromatography utilizing a specific inhibitor, $\text{trimethyl}\text{-p-}\text{aminophenyl}$ ammonium chloride, bound to Sepharose 4B. After a repeated chromatography acetylcholinesterase was found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunization of rabbits with acetylcholinesterase elicited the formation of an antiserum which gave single precipitin lines with the enzyme on immunodiffusion and rocket, crossed and immuno-electrophoreses. The purified enzyme had a specific activity of 418 units/mg protein. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of acetylcholinesterase with acetylthiocholine as substrate was $\text{1.5 × 10}{}^{\mathrm{?4}}\text{M}$. Isoelectric focusing of acetylcholinesterase in the presence of Triton X-100 and within the pH ranges of 3–10 and 3–6 exhibited a single peak of enzyme activity with a $\text{P}\text{I}$ of 4.8. The results of amino acid and carbohydrate analyses showed that acetylcholinesterase is a glycoprotein with a carbohydrate/protein weight ratio of 0.16 and glucose, galactose, mannose, glucosamine, galactosamine and sialic acid as the sugar components. The N-terminal amino acid was blocked. Lipid, phosphorus and fatty acid analyses indicated phosphatidylserine and cholesterol as the major lipid components of acetylcholinesterase. The apparent subunit molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol was 160 000 and in its presence, 80 000. The kinetic studies showed a competitive inhibition of acetylcholinesterase by its antibodies. Agglutination of human red cells by monospecific antiserum to acetylcholinesterase confirmed that the antigenic site(s) of the enzyme is localized on the outer surface of the erythrocyte membrane.     

pp60c-src Kinase is in chick and human embryonic tissues

The normal cellular protein pp60c-src is a tyrosine-specific protein kinase that is homologous to the transforming protein of Rous sarcoma virus (RSV) but its function is unknown. The expression of pp60c-src in chick and human embryonic tissues was monitored by the immune complex protein kinase assay, Western transfer analysis, and immunocytochemical staining at the light microscope level. pp60c-src kinase was expressed in the head and trunk regions of the chick embryo at all stages of development examined; however, expression increased significantly during the major period of organogenesis (Hamburger and Hamilton stages 21 to 32). Western transfer analysis showed that the amount of pp60c-src protein increased in parallel with the increase in kinase activity. Highest levels of pp60c-src kinase were present in the neural tube, brain, and heart of the stage 32 chick embryo. Lower levels of activity were found in eye, limb bud, and liver. Immunocytochemical staining of the neural tube region and heart of the chick confirmed the results of biochemical analysis and showed immunoreactive pp60c-src distributed throughout the neural tube and heart. The distribution of pp60c-src kinase in human fetal tissues was similar to that in the chick embryo; elevated levels of pp60c-src kinase were present in cerebral cortex, spinal cord, and heart, but all other tissues examined expressed some pp60c-src kinase. The results of our studies suggest that pp60c-src plays a fundamental role in an aspect of cellular metabolism that is particularly important in electrogenic tissues.     

Enzymatic assays of L-ornithine and L-delta 1-pyrroline-5-carboxylate in tissues, and orniathine-load test in human subjects

The efficiencies of estimates obtained from the direct linear plot (A. Cornish-Bowden and R. Eisenthal, 1978, Biochem. Biophys, Acta, 523, 268) are shown to be dependent on the spacing of substrate concentrations. When substrate values are harmonically spaced, the direct linear plot should not be used. The nonparametric confidence limits based on the direct linear plot are accurate in their confidence coefficient, but their efficiencies are shown to be dependent on substrate spacing. Harmonic spacing is, in general, a more efficient experimental design for estimating K_m than arithmetic spacing when the appropriate estimation methods are used. If assumptions about the error structure cannot be made, the best procedure for estimating K_m is to have harmonic spacing of substrate values and use weighted least squares for estimation. The most accurate and precise estimation of enzyme kinetic parameters requires knowledge of the error structure and utilization of the appropriate nonlinear regression.     

Fate of glucose in haemolymph of the American cockroach, Periplaneta americana

The rate of removal of high concentrations of glucose (10 μg/μl haemolymph) from haemolymph of adult male cockroaches, Periplaneta americana, was studied in normal and ligated insects. More than 50% of the injected glucose is removed from the haemolymph of normal insects within 20 min of injection. A period of rapid trehalose synthesis occurs during the initial 10 min following injection of glucose into the haemocoele, and this is succeeded by a period of glycogen synthesis. The results are discussed in terms of earlier observations on ‘stress-induced hypertrehalosemia’ and the possible involvement of a glycogenic agent.     

Phosphorylation of glycophorin A in membranes of intact human erythrocytes

The qualitative and quantitative contribution of glycophorin A phosphorylation to the general and specific pattern of membrane protein phosphorylation in intact erythrocytes pre-incubated with 32Pi was examined. Intense 32P-labeled bands at 88,000 and 38,000 Mr were identified as phosphorylated glycophorin A dimer and monomer respectively on the basis of several criteria. Quantitatively, phosphorylated glycophorin A dimer accounted for about 70% of 32P in the band 3 region. This value is at least three times that previously reported. The results of ancillary experiments involving selective extraction of ghosts in acidified chloroform/methanol solvents and electrophoresis in the presence of detergents make it unlikely that the 32P associated with glycophorin A was due to bound polyphosphoinositides.     

Water transport in a cluster of closely packed erythrocytes at subzero temperatures

A one-dimensional model has been developed to describe the kinetics of water transport in a cluster of closely packed cells. For the case of human red blood cells, the intracellular medium has been treated as an ideal, hydrated, nondilute multicomponent electrolyte solution. Results show that the volume flux of water out of the interior cells of the cluster lags behind that of the exterior cells. At any given temperature (or time), the amount of water retained within a cluster of closely packed cells of a given type exceeds (on an overall percentage basis) the amount of water retained within a single isolated cell of the same type. For a given cooling rate the probability of intracellular ice nucleation at any given temperature will therefore be greater for cells in the interior of a cluster, and the survival signature for a cell cluster should peak at a cooling rate which is less than the corresponding optimal value for a single, isolated cell. These results are consistent with experimental observations.     

Macrophage-mediated cytolysis off erythrocytes in the guinea pig. I. Activation by stimulators of the oxidative burst

Oxygen metabolites generated by macrophages may exert membrane injury to various cells. In this study reagents, which induce superoxide (O₂^?) and hydrogen peroxide (H₂O₂) production by paraffin oil elicited adherence purified guinea pig peritoneal macrophages (GPPM), were studied as to their potential to activate macrophage-mediated cytolysis (MMC) against allogeneic and autologous erythrocytes. Strong MMC reactions were activated by 12-O-tetra-decanoylphorbol-13-acetate (TPA), methylated TPA (4-O-MeTPA), opsonized zymosan, and out of six lectins tested, by wheat germ agglutinin (WGA) and concanavalin A (Con A). The cGMP elevators: sodium nitroprusside and sodium azide and the formyl-methionyl-type chemotactic peptides were ineffective. MMC activated by TPA, 4-O-MeTPA, WGA, and Con A was unaffected by colchicine and partially inhibited by cytochalasin B. TPA-activated MMC was abolished by diethyldithiocarbamate (DDC) (inhibitor of superoxide dismutase) and catalase, while WGA and Con A-activated MMC were only partially inhibited by DDC and unaffected by catalase.     

Fluorine NMR studies of fluorocinnamoylchymotrypsins

Three monofluorocinnamoylchymotrypsins have been examined at pH 4 by fluorine NMR spectroscopy. Protein-induced fluorine chemical shifts are quite large (~7 ppm) when fluorine is present at the para position but nearly zero for ortho fluorine. The shifts roughly parallel those observed in complexes formed between the enzyme and the analogous N-acetylfluorophenylalanines, suggesting a similarity in molecular environment for the aromatic ring in both systems. Little correlation is found, however, between the shifts for the acylenzymes and those of the corresponding enzyme-cinnamate complexes, indicating that the environment for the aromatic ring in the complexes is dissimilar from that experienced by the aromatic group in the acylated enzyme. Solvent isotope effects ($\text{H}{}_2\text{O}\text{D}{}_2\text{O}$) on the fluorine chemical shifts for the fluorocinnamoylchymotrypsins are small and downfield. Fluorine NMR observations suggest that the presence of the fluorocinnamoyl group greatly stabilizes the enzyme toward denaturation in 8 m urea.     

Induction of calcium-binding protein in organ-cultured chick intestine by fluoro analogs of vitamin D3

Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D₃ (D₃) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D₃) or in both the 1α and 25 positions (1α,25-F₂-D₃) were markedly more potent than vitamin D₃ itself although 1α,25-F₂-D₃ was only $\text{1}\text{7}\text{th}$ as potent as 1α-F-D₃. The 25-fluoro analog (25-F-D₃) was a very weak inducer; only $\text{1}\text{45}\text{th}$ as potent as vitamin D₃. The 25-fluoro analog of 1α-hydroxyvitamin D₃ (1α-OH-25-F-D₃) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24R-OH-25-F-D₃ was about 15 times more potent than 24R,25-(OH)₂-D₃. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F₂-25-OH-D₃ and 24,24-F₂-1α,25-(OH)₂-D₃ were about four times as potent as their nonfluorinated counterparts. The 24,24-F₂-1α,25-(OH)₂-D₃ is, therefore, the most potent vitamin D₃ analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D₃ metabolite, 1α,25-(OH)₂-D₃.     

The role of copper and quaternary structure on the conformational properties of Octopus vulgaris hemocyanin

Some structural properties of Octopus vulgaris hemocyanin have been investigated by fluorescence spectroscopy. The three-dimensional structure of Octopus hemocyanin is remarkably tight, resulting in a deep burial of almost all the tryptophyl residues of the protein. The hemocyanin conformation has been studied in the two main aggregation states (11 S, 50 S) of the protein, and with respect to the presence or absence of copper in the active site. Upon changing the pH of the solution, Octopus hemocyanin in the 50 S aggregation state can assume at least three different conformations. During the transition between each conformation the fluorescence quantum yield changes, but the environment of tryptophans does not change. Dissociation of the protein from 50 S to 11 S strongly enhances its susceptibility toward denaturating agents such as pH or temperature, and modifies the effects of fluorescence quenchers such as acrylamide. Moreover, these effects are more pronounced when copper is removed from the active site. A comparative analysis of the results shows that the subunit-subunit interactions exerted within the 50 S species are more important in the maintenance of the conformational stability than the copper ions present in the active sites. This behavior can be accounted for by the large amount of Ca(II) ions linked to 50 S hemocyanin.     

Interaction between calcium and ligand-binding sites of the purified acetylcholine receptor studied by use of a fluorescent lanthanide

The acetylcholine receptor isolated from $\text{Torpedo ocellata}$ binds about 10 moles of a fluorescent lanthanide, terbium, per mole α-bungarotoxin-binding site, a process which is accompanied by a fluorescence enhancement (λexcitation 295 nm, λemission 546 nm) which allows detection of receptor-Tb³⁺ complexes at μM concentrations. In presence of calcium two types of terbium-binding site are revealed, both with terbium dissociation constants of 18 ± 0.5 μM. About 60% of the sites bind calcium with an apparent dissociation constant of 1.1 ± 0.1 mM. Sites which interact with calcium also interact with activators of neural transmission, carbamylcholine and decamethonium, but not with the inhibitors, d-tubocurarine and α-bungarotoxin. Whether the displacement of calcium by chemical mediators is directly responsible for activator-induced changes in ion permeability of neural membranes is an important question raised by our experiments. The results show that fluorescent lanthanides can be an important tool in such studies.