Peripheral proteins of human erythrocytes

Water soluble, nonglycosylated proteins have been extracted from human erythrocyte membranes by two different methods and characterized immunochemically and by PAGE. The spectrin peripheral protein complex (PAGE bands 1 + 2) has been equated with two antigens of intermediate mobility in immunoelectrophoretic analysis of crude spectrin developed with antiserum to bands 1 + 2 purified by elution from gels. Nonspectrin proteins, including catalase, remain in close association with isolated membranes, and display solubility properties similar to those of spectrin. Along with spectrin, they may also function in the intact cell as peripheral proteins.     

NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation.

A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH. These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate.     

Mechanism of selective release of membrane proteins from human erythrocytes in the presence of liposomes

Incubation of erythrocytes with liposomes results in the release of shed vesicles rich in glycosyl-phosphatidylinositol (GPI)-anchored proteins but poor in transmembranous proteins. We investigated the mechanisms of membrane protein polarization by examining the effect of the interaction between spectrin and membrane proteins on the release of a transmembranous protein, band 3, and a GPI-anchored protein, acetylcholinesterase (AChE), from erythrocyte ghosts. Polymerization of spectrin resulted in a 30-fold decrease in the released amount of band 3 per constant amount of shed vesicles but did not affect the amount of released AChE per constant amount of shed vesicles. On the other hand, the amount of released band 3 per constant amount of shed vesicles increased by cleaving the cytoplasmic part of band 3. Our results first demonstrated that the diffusibility of membrane proteins determined by steric hindrance between membrane proteins and protein mesh primarily determines the ease of localization of membrane proteins into shed vesicles. Taken together with the recent biophysical studies, we built a "fence selection model" that retrograding spectrin mesh sweeps diffusing band 3 molecules from the tip of the membrane crenated area toward the entry of the crenated area, but not AChE molecules. Our study describes a novel method for isolation of a large number of vesicles containing special and intact membrane proteins from cells not by using detergents or organic solvents, but by utilizing the fence effect between the cytoskeleton and membrane proteins.     

Degradation of transmembrane proteins in Ca2+-enriched human erythrocytes. An immunochemical study

Apart from causing the formation of gamma-glutamyl-epsilon-lysine cross-linked polymers, exposure of human erythrocytes to Ca2+ and ionophore A23187 leads to a breakdown of the two major transmembrane proteins, i.e. the anion-transporting band 3 and glycophorin. This apparently proteolytic phenomenon was examined by crossed immunoelectrophoretic techniques. The main product of the cleavage of band 3 had a chain weight of about 55,000 and showed good precipitation with the antibody raised against the intact protein. The degradation of glycophorin was more rapid and, when complete, gave rise to small fragments which were barely precipitated with antiglycophorin antibody. Incubation of the cells with pepstatin or N-ethylmaleimide prior to and during Ca2+ loading prevented the breakdown of both transmembrane proteins. Histamine, a competitive inhibitor of the transglutaminase-catalyzed formation of gamma-glutamyl-epsilon-lysine cross-links in Ca2+-enriched erythrocytes, also provided some protection, suggesting that the breakdown of the two transmembrane proteins might in some manner be related to the transglutaminase-dependent polymerization process. Pathophysiological implications of the proteolytic event, which would distort the normal interaction of membrane proteins with the cytoskeleton, are discussed.     

Adenine-induced hypoxanthine release from IMP-enriched human erythrocytes

Adenine uptake and hypoxanthine release by IMP-enriched human erythrocytes has been studied. The presence of IMP within the erythrocytes leads to an increase in the rate of adenine incorporation. Adenine is taken up by IMP-enriched erythrocytes as AMP, even when intracellular 5-phoshorobosyl-1-pyrophosphate concentration is undetectable and too low to allow IMP synthesis from hypoxanthine. During adenine uptake and AMP synthesis, hypoxanthine is released by the cells. The possibility that 5-phosphoribosyl-1-pyrophosphate, necessary for AMP synthesis, is formed through the hypoxanthine guanine phosphoribosyltransferese-catalyzed IMP pyrophosphorolysis is considered.     

In vivo operation of the pentose phosphate pathway in frog oocytes is limited by NADP+ availability

Evolution of CO2 from labelled glucose microinjected into frog oocytes in vivo may be ascribed to the pentose-P pathway, as measured by radioactive CO2 production from [1-(14)C] and [6-(14)C]glucose. Coinjection of NADP+ and [14C]glucose significantly stimulated 14CO2 production. The effect depends on the amount of NADP+ injected, half maximal stimulation being obtained at 0.13 mM. The increase in CO2 production was also observed with microinjected glucose-1-P, glucose-6-P or fructose-6-P used as substrates. Phenazine methosulfate, mimicked the effects of NADP+. A high NADPH/NADP+ ratio of 4.3 was found in the cells, the intracellular concentration of NADP+ being 19 microM.     

Adrenodoxin reductase. Properties of the complexes of reduced enzyme with NADP+ and NADPH.

Anaerobic reduction of the flavoprotein adrenodoxin reductase with NADPH yields a spectrum with long wavelength absorbance, 750 nm and higher. No EPR signal is observed. This spectrum is produced by titration of oxidized adrenodoxin reductase with NADPH, or of dithionite-reduced adrenodoxin reductase with NADP+. Both titrations yield a sharp endpoint at 1 NADP(H) added per flavin. Reduction with other reductants, including dithionite, excess NADH, and catalytic NADP+ with an NADPH generating system, yields a typical fully reduced flavin spectrum, without long wavelength absorbance. The species formed on NADPH reduction appears to be a two-electron-containing complex, with a low dissociation constant, between reduced adrenodoxin reductase and NADP+, designated ARH2-NADP+. Titration of dithionite-reduced adrenodoxin reductase with NADPH also produces a distinctive spectrum, with a sharp endpoint at 1 NADPH added per reduced flavin, indicating formation of a four-electron-containing complex between reduced adrenodoxin reductase and NADPH. Titration of adrenodoxin reductase with NADH, instead of NADPH, provides a curved titration plot rather than the sharp break seen with NADPH, and permits calculation of a potential for the AR/ARH2 couple of -0.291 V, close to that of NAD(P)H (-0.316 V). Oxidized adrenodoxin reductase binds NADP+ much more weakly (Kdiss=1.4 X 10(-5) M) than does reduced adrenodoxin reductase, with a single binding site. The preferential binding of NADP+ to reduced enzyme permits prediction of a more positive oxidation-reduction potential of the flavoprotein in the presence of NADP+; a change of about + 0.1 V has been demonstrated by titration with safranine T. From this alteration in potential, a Kdiss of 1.0 X 10(-8) M for binding of NADP+ to reduced adrenodoxin reductase is calculated. It is concluded that the strong binding of NADP+ to reduced adrenodoxin reductase provides the thermodynamic driving force for formation of a fully reduced flavoprotein form under conditions wherein incomplete reduction would otherwise be expected. Stopped flow studies demonstrate that reduction of adrenodoxin reductase by equimolar NADPH to form the ARH2-NADP+ complex is first order (k=28 s-1). When a large excess of NADPH is used, a second apparently first order process is observed (k=4.25 s-1), which is interpreted as replacement of NADPH for NADP+ in the ARH2-NADP+ complex. Comparison of these rate constants to catalytic flavin turnover numbers for reduction of various oxidants by NADPH, suggests an ordered sequential mechanism in which reduction of oxidant is accomplished by the ARH2-NADP+ complex, followed by dissociation of NADP+. The absolute dependence of NADPH-cytochrome c reduction on both adrenodoxin reductase and adrenodoxin is confirmed...     

Two-dimensional map of membrane proteins from human erythrocytes

Human erythrocyte membrane proteins were analyzed by a modified two-dimensional electrophoresis performed according to O'Farrell. This method was used to construct a two-dimensional map of human erythrocyte membrane proteins. The map plotted in the coordinates "relative molecular mass versus relative electrophoretic mobility during IEF" was used for the characterization of 189 proteins. The position of major membrane proteins in the map was determined on the basis of their Mr, pI as well as literature data. Carboanhydrase was positioned by coelectrophoresis. A comparative analysis of erythrocyte membrane and cytosol preparations by two-dimensional protein mapping revealed that some of erythrocyte proteins have dual localization.     

Ferredoxin:NADP+ oxidoreductase. Equilibria in binary and ternary complexes with NADP+ and ferredoxin

Ferredoxin:NADP+ oxidoreductase (ferredoxin: NADP+ reductase, EC 1.18.1.2) was shown to form a ternary complex with its substrates ferredoxin (Fd) and NADP(H), but the ternary complex was less stable than the separate binary complexes. Kd for oxidized binary Fd-ferredoxin NADP+ reductase complex was less than 50 nM; Kd(Fd) increased with NADP+ concentration, approaching 0.5-0.6 microM when the flavoprotein was saturated with NADP+ K(NADP+) also increased from about 14 microM to about 310 microM, on addition of excess Fd. The changes in Kd were consistent with negative cooperativity between the associations of Fd and NADP+ and with our unpublished observations which suggest that product dissociation is rate-limiting in the reaction mechanism. Similar interference in binding was observed in more reduced states; NADPH released much ferredoxin:NADP+ reductase from Fd-Sepharose whether the proteins were initially oxidized or reduced. Complexation between Fd and ferredoxin: NADP+ reductase was found to shield each center from paramagnetic probes; charge specificity suggested that the active sites of Fd and ferredoxin:NADP+ reductase were, respectively, negatively and positively charged.     

Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli

Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation. FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor. We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C. The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin. Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.     

Hypoxia reduced upper thermal limits causing cellular and nuclear abnormalities of erythrocytes in Nile tilapia,Oreochromis niloticus

Global warming is a threat across the world that leads to estimates of the upper thermal limits of ectothermic species. Increased water temperature up-regulates oxygen consumption and metabolic rates, and alters the physiological processes. In this study, we identified the critical thermal maxima (CTmax) and physiological responses under normoxia and hypoxia in Nile tilapia, Oreochromis niloticus. CTmax was 41.25 °C under hypoxia and 44.50 °C under normoxia. Compared to normoxia, lower values of hemoglobin (Hb) and red blood cells (RBCs) were observed at the CTmax under hypoxia. In contrast, higher values of white blood cells (WBCs) and blood glucose (Glu) levels were observed at the CTmax under hypoxia. Consequently, higher frequencies of micronucleus, cellular and nuclear abnormalities of erythrocytes were observed at the CTmax under hypoxia. These results suggest that high temperature tolerance and subsequent physiology are significantly affected by the oxygen supply in Nile tilapia. As climate vulnerability is intensifying day by day, this data will be helpful in successful management practice for the aquatic environment having low oxygen content.     

Furosemide-sensitive Na+-K+ cotransport and cellular metabolism in human erythrocytes

Metabolic depletion of human red cells with 2-deoxy-D-glucose in the presence of EGTA decreased ATP to about 4% of the initial value and increased total ouabain- and furosemide-resistant Na+ and K+ effluxes by 20% and 100%, respectively, and furosemide-sensitive Na+ and K+ effluxes by 100% and 60%, respectively. When ATP was restored, all the components of Na+ and K+ fluxes measured returned to baseline levels suggesting a metabolic dependence.     

Enzymatic basis for the Ca2+-induced cross-linking of membrane proteins in intact human erythrocytes.

The accumulation of Ca2+ ions in intact human erythrocytes leads to the production of membrane protein polymers larger than spectrin. The polymer has a heterogeneous size distribution and is rich in gamma-glutamyl-epsilon-lysine cross-links. Isolation of this isodipeptide, in amounts as high as 6 mol/10(5) g of protein, confirms the idea [Lorand L., Weissmann, L.B., Epel, D.L., and Bruner-Lorand, J. (1976), Proc. Natl. Acad. Sci. U.S.A. 73, 4479] that the Ca2+-induced membrane protein polymerization is mediated by transglutaminase. Formation of the polymer in the intact cells is inhibited by the addition of small, water-soluble primary amines. Inasmuch as these amines are known to prevent the Ca2+-dependent loss of deformability of the membrane, it is suggested that transglutaminase-catalyzed cross-linking may be a biochemical cause of irreversible membrane stiffening.     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

Sulphydryl groups involved in Na+-Li+ exchange in human erythrocytes

Oxidative stress causes cellular injury that is thought to be due to increased cytosolic cation levels. Disturbances of a variety of mechanisms which normally maintain intracellular anion/cation homeostasis, occur during oxidative stress. Reactivity of the SH- groups essential for oubain-resistant Na(+)-Li(+) exchange by N-ethylmaleimide (NEM) and selenite was studied in human erythrocytes. In addition, the reactivity of the substances on SH- groups and Li(+) influx have been studied as a function of pH of the medium. The results show that NEM induces an irreversible inhibition of Li(+) influx. It diminishes progressively with the increasing pH of the medium. Whereas we obtain increasing intracellular Li(+) concentration with the rising selenite concentration in the medium. The maximum effect with this substance is reached at about pH 8.0. We can state that the -SH reagents (NEM and selenite) studied behave differently: NEM inhibits Li(+) influx by modifying the essential SH-groups of the membrane proteins in such a way that the exchange is reduced, whereas it maintains the Na(+) permeability almost unaltered. The slight increase in intracellular Na(+) induced by selenite suggests that the oxidative changes in the intracellular sulphydryl groups may constitute an important mechanism for the regulation of the intracellular cations.