Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

The penetration of local anesthetics into the red blood cell membrane as studied by fluorescence quenching.

The local anesthetics procaine and tetracaine were found to quench the fluorescence of the probes N-octadecyl naphthyl-2-amine 6-sulfonic acid and 12-(9-anthroyl)stearic acid in the presence of erythrocyte membranes. This quenching was shown to be due to the aromatic amine of the procaine and tetracaine molecules. Lidocaine, an active anesthetic that does not contain an aromatic amine in the same position as does procaine and tetracaine did not quench either of the fluorophores. The preferential quenching of the fluorescent probes by procaine and tetracaine indicated a greater accessibility of tetracaine than of procaine to the hydrocarbon region of the membrane and a greater accessibility of procaine than of tetracaine at the membrane's surface. The addition of calcium was found to reverse the quenching of 12-(9-anthroyl)stearic acid by tetracaine in the presence of red cell membranes.     

Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes

ATP stimulates Na transport into inside-out vesicles (IOVs) made from human red cell membranes; strophanthidin inhibits the ATP-stimulated transport. The substrates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (glycolytic enzymes bound to the cytoplasmic surface of the red cell membrane) also stimulate Na transport into IOVs without added ATP. The elution of GAPDH from the membranes prevents the stimulation by the substrates, but not by exogenous ATP. Hexokinase plus glucose (agents that promote breakdown of ATP) prevent stimulation of Na transport by exogenous ATP but not by the substrates for GAPDH and PGK. [32P]orthophosphate is incorporated into a membrane-bound organic phosphate compound shown chromatographically to be ATP. The level of membrane-bound ATP is decreased when Na is added, and this decrease is inhibited by strophanthidin. When further synthesis of [32P]ATP is blocked by the addition of unlabeled orthophosphate, all of the membrane-bound [32P]ATP is dissipated by the addition of Na. From these observations it was concluded that membrane-bound glycolytic enzymes synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.     

Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.     

Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.

The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.     

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. ⁵⁴Mn²⁺ uptake occurred on stimulation but at a lower rate than ⁴⁵Ca²⁺ uptake. There was no delay of ⁵⁴Mn²⁺ uptake upon stimulation and ⁵⁴Mn²⁺ uptake was considerably prolonged compared to ⁴⁵Ca²⁺ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd²⁺>Mn²⁺>Ca²⁺>Sr²⁺.     

Effects of divalent metal ions on the calcium pump and membrane phosphorylation in human red cells

In inside-out red cell membrane vesicles ATP-dependent calcium transport is activated by the divalent metal ions Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Fe²⁺. This activation is based on the formation of Me²⁺-ATP complexes which can serve as energy-donor substrates for the calcium pump, and probably, satisfy the requirement for free Me²⁺ in this transport process. Higher Me²⁺ concentrations inhibit calcium transport with various efficiencies. Mn²⁺ directly competes with Ca²⁺ at the transport site, while other divalent metal ions investigated have no such effect. The formation of the hydroxylamine-sensitive phosphorylated intermediate (EP) of the red cell membrane calcium pump from [γ-³²P]ATP is induced by Ca²⁺ while rapid dephosphorylation requires the presence of Mg²⁺. At higher concentrations Mn²⁺ and Ni²⁺ inhibit predominantly the formation of EP, while Co²⁺ and Fe²⁺ block dephosphorylation. The possible sites and nature of the divalent metal interactions with the red cell calcium pump are discussed. Hydroxylamine-insensitive membrane phosphorylation in inside-out vesicles from [γ-³²P]ATP is significantly stimulated by Mn²⁺ and Co²⁺, as compared to that produced by Mg²⁺, Fe²⁺ and Ni²⁺. Part of this labelling is found in phospholipids, especially in phosphatidylinositol. The results presented for the metal dependency of protein and lipid phosphorylation in red cell membranes may help in the characterization of ATP consumptions directly related to the calcium pump and those involved in various regulatory processes.     

Side-specific effects of sodium on (Na,K)-ATPase. Studies with inside-out red cell membrane vesicles.

Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase have been studied using low concentrations of [gamma-32P]ATP (less than or equal to 0.1 microM). Phosphorylation is stimulated by Na+ at the cytoplasmic membrane surface (extravesicular Na+) alone and not by Na+ at the external surface (intravesicular Na+). At 37 degrees C, external Na+ (less than or equal to 10 mM) does, however, increase the steady state level (approximately 2 1/2-fold) of phosphoenzyme above that observed with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0 degrees C. As Na+ at the cytoplasmic side is decreased to very low levels (less than or equal to 0.2 mM) several kinetic changes are observed: (i) the apparent turnover of phosphoenzyme (ratio Na+-ATP-ase/phosphoenzyme level) is markedly increased (approximately 3-fold, (ii) Rbext sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced, and (iii) the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. These results are compatible with a reaction pathway involving a transition from one form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+. It is suggested also that an alternate reaction pathway for Na+-ATPase occurs at very low cytoplasmic Na+, one via hydrolysis of E1-P and not associated with Na+ translocation.     

Evidence for the mobilization of cellular calcium by prostacyclin in cat adrenocortical cells: The effect of TMB-8

Stimulation of steroid production by isolated cat adrenocortical cells is partially dependent upon the presence of extracellular Ca²⁺ when elicited by prostacyclin (PGI₂) and completely dependent upon extracellular Ca²⁺ when elicited by corticotropin. TMB-8, an intracellular Ca²⁺ antagonist, completely blocked PGI₂-evoked steroid output in the absence of external Ca²⁺; this inhibition was partially reversed by the addition of Ca²⁺. The increase in secretion caused by corticotropin or PGI₂ in the presence of Ca²⁺ was also reduced in a dose-dependent manner by TMB-8. The steroidogenic action of pregnenolone, which is induced by a Ca²⁺ independent mechanism, was not blocked by TMB-8, either in the presence or absence of Ca²⁺. Corticotropin significantly potentiated the Ca²⁺-independent aspect of PGI₂ action. These studies provide evidence for an internal, PGI₂-sensitive Ca²⁺ store in cat adrenocortical cells.     

Transport and control of Ca2+ by pigeon erythrocytes. II. Evidence against a simple feedback control of cell Ca2+ and evidence for the involvement of more than one pool

Pigeon erythrocytes did not behave as expected from simple feedback mechanisms. The pool size for exchangeable cell Ca2+ was approximately proportional to the A23187-induced apparent 45Ca2+ influx ("J(in,app)") from 0.4 to 14 mumoles/min X l cell water at 184 microM external [Ca2+]. From earlier data, total cell 45Ca2+ was approximately proportional to J(in,app) from 10 to 120 mumoles/l X min. Thus there was no influx range where cell 45Ca2+ was held approximately constant. External [Ca2+] affected Ca2+ pool size independently of its effect of J(in,app). Trifluoperazine did not increase cell 45Ca2+ with or without A23187. In the presence of A23187, 45Ca2+ entered a pool early in the incubation which later became inaccessible to 45Ca2+ entry and exit. Lysolecithin addition produced an abrupt rise in cell 45Ca2+, much of which occupied a pool that quickly became inaccessible. The increased 45Ca2+ influx induced by lysolecithin dropped quickly and markedly with time. It is hard to explain inaccessible pool(s), especially in the presence of A23187 by membrane-bounded compartments. We suggest that nonexchangeable 45Ca2+ might be held by an energy-dependent binding protein(s).     

The effect of in vitro and in vivo cellular aging on the active calcium transport in human inside-out red cell membrane vesicles

Modelling of the in vivo and in vitro aging processes in the human red cell has stressed the following features of the active calcium uptake by inside-out vesicles: 1) it is higher in the outdated, in vitro aged, than in the fresh red cell (p less than 0.0005), and in the densest, in vivo aged fraction than in the lightest, young fraction (p = 0.08); 2) it increases following stimulation by excess calmodulin to values that are not significantly different; 3) it decreases to the same value in the absence of endogenous calmodulin and inhibitor, with and without exogenous calmodulin; 4) it is the target of a non-competitive inhibition, that is stronger in the fresh than in the outdated red cell. We conclude that the aging process does not involve neither membrane Ca-ATPase nor calmodulin, but rather the interaction of the calcium pump with the inhibitor of Ca-ATPase.     

Sidedness of (sodium, potassium)-adenosine triphosphate of inside-out red cell membrane vesicles. Interactions with potassium.

Inside-out membrane vesicles of human red cells, prepared according to the method of Steck et al. (1970) Science 168, 255-257) have sufficiently low cation permeability to allow the examination of the side-specific interactions of ligands with the asymmetric sodium pump complex. In accordance with the known properties of the pump in intact cells the following results were observed: (a) ATP-dependent sodium influx and (b) maximal (sodium, potassium)-ATPase with K+ present inside the vesicles with larger than or equal to 20 micronM ATP. With much lower [ATP], K+ inhibited sodium-activated ATPase. K+ was inhibitory at either surface. Inhibition was different on the two sides since cytoplasmic (extravesicular) Na+ counteracted inhibition by cytoplasmic (extravesicular) K+ but not inhibition by K+ at the plasma or external membrane surface, i.e. intravesicular K+. A decrease in the steady state level of the phosphenzyme intermediate of sodium-activated ATPase was caused also by K+ at either surface. The effect of cytoplasmic K+ is compatible with its competitive inhibition of activation of phosphorylation of the enzyme by cytoplasmic Na+. At 37 degrees, the inhibitory effect of external K+ is due to interaction with the phosphoenzyme to form a stable complex of K+ with the dephosphenzyme resulting in a decreased overall reaction rate but increased turnover of the phosphoenzyme (E-P + K leads to EK + Pi). At 0 degree, external K+ inhibits by interacting with the unphosphorylated enzyme to form an occluded enzyme-K complex. This results in a decreased overall rate but relatively small change in apparent turnover of the phosphoenzyme. At 0 degree, but not at 37 degrees, external Na+ counteracted the inhibitory effects of external K+.     

Effects of freeze-preservation on some pollen enzymes: II. Freezing and freeze-drying stresses

The isozymes of lily and corn pollen esterases and acid phosphatase were studied in relation to freeze-drying and vacuum-drying. Fresh samples of Lilium longiflorum L. and Zea mays L. pollen were frozen at rates ranging between 200 and 100 °C/min and freeze-dried at temperatures from 0 to ?70 °C for approximately 48 to 70 hr. Freeze-dried samples were rehydrated slowly (10% relative humidity) and rapidly (90% relative humidity). Vacuum-drying was performed at room temperature (22 °C).Soluble pollen enzymes were analyzed by disc electrophoresis on polyacrylamide gels stained with substrate specific reagents. The stained gels were evaluated by densitometry for migration rate, isozyme pattern, and relative activity. The numerical data generated in this manner were then statistically analyzed.The following conclusions resulted from this study: (i) freeze-drying and freeze-thawing treatments were comparable except for corn esterases; (ii) freeze-drying induced alterations in enzyme activity except for corn acid phosphatase; (iii) the freezing rate, the final freezing temperature, and exposure to various relative humidities produced few changes in freeze-dried material; (iv) freeze-drying was less detrimental than vacuum-drying to the enzyme characteristics of corn; (v) freeze-drying yielded higher viabilities than vacuum-drying; and (vi) acid phosphatase alterations appeared to be related to pollen viability in most cases.     

Effects of 2-formylpyridine monothiosemicarbazonato copper II on red cell components

2-Formylpyridine monothiosemicarbazonato copper II (CuL⁺) is readily taken up by red cells and is initially bound to glutathione and hemoglobin. Glutathione was depleted within 5 hr of incubation, presumably by oxidation mediated by CuL⁺ and O₂ with concomittant generation of toxic oxygen species. Cupric ion was slowly transferred from CuL⁺ to hemoglobin within about 7 hr, and hemoglobin was oxidized until the major form prevailing after 10 hr was α₂β₂⁺. Little increase in hemolysis due to addition of CuL⁺ dissolved in the radical scavenger dimethyl sulfoxide was observed with prolonged incubation. Strong inhibition of red cell hexokinase by CuL⁺ was observed when the enzymes in red cell lysates and hemoglobin-free red cell lysates were examined. CuL⁺ was also an effective inhibitor of yeast hexokinase. However, the inhibitory effect of CuL ⁺ within the red cells was less pronounced. It is suggested that even though intracellular accumulation of CuL ⁺ creates an oxidizing environment and is potentially capable of inhibiting thiol enzymes such as hexokinase, protective effects are exerted in the red cell by the presence of hemoglobin, of radical scavengers, and of high levels of enzymes that detoxify toxic oxygen species. Address reprint requests to Dr. W.E. Antholine, Department of Radiology, or Dr. F. Taketa, Department of Bio