Hemolysis Of Red Cells Research Articles

Different effects of thiol and nonthiol ace inhibitors on copper-induced lipid and protein oxidative modification

Differences among angiotensin-converting enzyme inhibitors (ACEI) in scavenging reactive oxygen species were described and mainly attributed to the presence or absence of a thiol group. Plasma constituents and red cells are known targets for oxidative damage. Transition metals, like copper, are well known catalizers of free radical generation. In the present study we compared the abilities of captopril (a thiol ACEI), enalaprilat, and lisinopril (two nonthiol ACEI) for inhibiting copper-induced thiobarbituric acid reactive substances (TBARS) formation and fluorescence generation in whole human plasma and low-density lipoprotein. The effects of those ACEI on copper/hydrogen peroxide-induced fluorescence development and electrophoretic mobility modification in albumin and on copper-induced TBARS formation and hemolysis in human red cells were also compared. Captopril was more effective than the two nonthiol ACEI in inhibiting plasma and LDL lipid peroxidation, but it was ineffective in inhibiting the albumin oxidative modification that was moderately inhibited by enalaprilat and lisinopril. On the contrary, the inhibitory effects of the three ACEI on copper-induced lipid peroxidation and hemolysis in red cell suspensions were more uniform. This as yet unreported red cell protective effect may deserve pharmacological evaluation. Our results show that captopril is a more effective antioxidant than the nonthiol ACEI in some systems. However, the nonthiol ACEI also have the ability to partially protect some targets against oxidative damage. These observations suggest that the presence of a thiol group in the ACEI structure is not the only determinant for the antioxidant properties

Oxazolidonylethyl adducts to hemoglobin and DNA following nornitrogen mustard exposure

Formation of adducts to hemoglobin (Hb) and DNA of nornitrogen mustard (NNM) was studied with the aim of developing a method for monitoring exposure to NNM. Adducts to N-terminal valines in Hb were studied by the N-alkyl Edman method using pentafluorophenyl isothiocyanate (PFPITC) as the derivatizing reagent. In preliminary studies five major Hb adducts were shown to be formed in reaction of NNM with red cell hemolysate in vitro. Following treatment with PFPITC three of these were found to be pentafluorophenylthiohydantoins (PFPTHs) of N-alkylated valines and the fourth probably originates from NNM esters in which PFPITC had reacted with the nitrogen of N-chloroethylaminoethyl. A PFPTH was found to originate from N-2-(3-oxazolidonyl)ethylvaline, Val-OZ. Val-OZ is formed in reaction, with ring closure to oxazolidone, of CO 2 with the 2-chloroethylamino group in the primary Nvaline- N adduct. Besides a few other adducts, Val-OZ was also observed in mouse Hb following injection of NNM, and also after injection of cyclophosphamide. Following reaction in vitro of NNM with DNA, three major adducts to guanine- N-7 were observed; one of them, 7-[ N′-(2-chloroethyl)-2-aminoethyl]-guanine (NNMCl), was converted by carbonate to 7-[2-(3-oxazolidonyl)ethyl]guanine (Gua-OZ). In mice treated with NNM, Gua-OZ was the only DNA adduct observed. Val-OZ is a chemically stable Hb adduct, potentially useful for monitoring exposures to NNM and cyclophosphamide.

In vivo anti-CD3-driven cell activation. Cellular source of induced tumor necrosis factor, interleukin-1 beta, and interleukin-6.

In vivo anti-CD3-driven cell activation. Cellular source of induced tumor necrosis factor, interleukin-1 beta, and interleukin-6.

Effects of stonustoxin (lethal factor from Synanceja horrida venom) on platelet aggregation

Stonustoxin (SNTX) is a 148,000 mol. wt lethal factor isolated from Synanceja horrida venom. It induces species-restricted red cell haemolysis which correlates with its effects on platelet aggregation in whole blood. SNTX induced a concentration-dependent and irreversible platelet aggregation in rabbit or rat but not in human or mouse whole blood. The degree of haemolysis and platelet aggregation induced by SNTX in rabbit or rat whole blood were concentration dependent. At concentrations of SNTX where only slight or no haemolysis was observed, no platelet aggregation occurred. Although SNTX itself could not induce platelet aggregation in rabbit or rat platelet-rich plasma (PRP), it had biphasic effects on collagenor ADP-induced platelet aggregation in PRP. It inhibited collagen- or ADP-induced platelet aggregation at high concentrations (0.08-0.8 μg/ml) but the response was potentiated by lower stonustoxin concentrations (0.008-0.016 μg/ml). The inhibition of collagen- or ADP-induced platelet aggregation might be due to the lytic activity of SNTX on the platelets.

Effects of exercise on erythrocytes in normal and splenectomised Thoroughbred horses

SummaryChanges in erythrocyte quality during exercise were determined in 3 control (Group C) and 3 splenectomised (Group 5) horses that performed an incremental exercise test on a treadmill until the point of fatigue. Venous blood samples were drawn before the exercise test and immediately after warming‐up and incremental exercise test. Incremental exercise increased erythrocyte count and haemoglobin concentration by 46 and 37% respectively in control horses and by 15 and 17% respectively inGroup Sanimals. Packed cell volume (PCV) increased by 48% in controls but there was no change inGroup Sanimals. Warming‐up decreased mean cell volume (MCV), mean cell Hb (MCH) and mean cell Hb concentration (MCHC) by 9, 19 and 7% in control horses. Incremental exercise restored MCV and MCHC but MCH remained lower than the resting level. InGroup Shorses, warming‐up and incremental exercise decreased MCV by 6 and 9% respectively, did not change MCH, but increased MCHC by 4 and 15% respectively. Incremental exercise increased erythrocyte density (ED) inGroup Cbut not inGroup Shorses. Osmotic fragility of erythrocytes measured as the red cell haemolysis rates in 0.56% NaCl (HL) were significantly higher inGroup Cthan inGroup Shorses regardless of exercise. Warming‐up decreased HL by 7% inGroup Cvs 19% inGroup Shorses, but exercise increased HL by 26% inGroup Cvs 38% inGroup Shorses. These results suggest that: 1) exercise causes shrinking of erythrocytes; 2) erythrocyte indices are dependent on the intensity of the exercise; 3) release of erythrocytes from the spleen is associated with an increase in osmotic fragility; and 4) exercise changes osmotic fragility regardless of the release of erythrocytes from the spleen into circulation.

The pharmacological manipulation of fetal haemoglobin: trials using hydroxyurea and recombinant human erythropoietin.

Hydroxyurea (HU) and recombinant human erythropoietin (rHuEpo) have been used in several studies to elevate Hb F level in sickle cell disease (SCD) patients and hence to ameliorate the clinical presentations of the disease. The treatment protocol and doses have varied in the different studies. We studied the effects of HU+rHuEpo combination therapy in sickle cell anaemia (SCA patients) to investigate the Hb F manipulation and hence treatment of SCA. Six patients with severe SCA were selected for treatment with HU (20-25 mg/kg body weight) and rHuEpo (400-800 U/kg body weight) combination therapy for 4 weeks followed by HU (20-25 mg/kg body weight) maintenance therapy for 6 months to 1 year. Iron and folic acid were administered during HU+rHuEpo treatment. Signs, symptoms and complications were recorded to obtain the severity index. Only patients with a severity index > or = 6 were included in the study. Haematological and biochemical parameter values, Hb A2, Hb F, Hb F distribution, Hb F cells, bilirubin level and reticulocyte count were assessed at least on 2-3 occasions prior to initiation of the therapy protocol to establish baseline values. During the treatment period, the clinical presentations were monitored and the estimation of the laboratory parameters was carried out every 4-8 weeks. The results of these parameters during HU and rHuEpo combination therapy and HU maintenance therapy were compared with baseline values using paired t test. The elevation in the level of Hb F, Hb F cells, total haemoglobin, red cell count and MCV were significant (p < 0.005), while reticulocyte count and total bilirubin were significantly decreased (p < 0.05). Each patient showed an individual pattern of Hb F elevation. The increase in Hb F level was correlated with the haematological and biochemical parameters using the General Linear Model Programme of Statistical Analysis System. In general, the clinical presentation improved as Hb F level increased in each patient. In addition, the positive correlation with the haematological parameters and negative correlation with reticulocytes and total bilirubin confirmed the beneficial effect of elevated Hb F level on reducing red cell haemolysis. No correlation could be demonstrated between the pretreatment Hb F level and the increase in Hb F during the treatment period. Daily doses of HU with a single intravenous rHuEpo and iron supplementation elevate Hb F and Hb F cells in SCA patients. The Hb F level can be maintained high on HU therapy alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipid peroxidation parameters and activities of lysosomal enzymes in diabetics

The processes of lipid peroxidation and activities of lysosomal enzymes were studied in 56 patients with type I and II diabetes mellitus. The rate of lipid peroxidation of red cell membranes was assessed from the activities of enzymatic (NADPH-dependent) and nonenzymatic (ascorbate-dependent) . lipid peroxidation, from accumulation of acylhydroperoxides, in- termolecular joints, and from spontaneous red cell hemolysis. Activities of lysosomal enzymes (cathepsines, acid DNAse, and P-galactosidase) were measured in leukoconcentrate. The activity of enzymatic system of lipid peroxidation and acylhydroperoxide content in red cell membranes were found increased. In parallel with this, a deficiency in leukocytic lysosomes of P-galactosidase and DNAse was revealed. The detected metabolic disturbances may be regarded as one of the pathogenetic mechanisms of development of diabetic angiopathies. A relationship was revealed between changes in lipid peroxidation parameters and activities of lysosomal enzymes, on the one hand, and diabetes mellitus type and duration, on the other.

Oxidants Generated by the Myeloperoxidase-Halide System Activate the Fifth Component of Human Complement, C5

Hypochlorite and taurine chloramine (T-NCl) convert native fifth component of human complement (C5) to an activated state. This is evident from loss of functional properties of native C5 and acquisition of a binding site for C6 which is characteristic of C5b, the physiological activation fragment of C5. The complex of activated C5 with C6 is capable of combining with the components C7, C8, and C9 forming the cytotoxic terminal complement complex C5-9. The activation of C5 and its assembly with the late reacting complement components has been detected by reactive lysis, i. e. hemolysis of unsensitized red cells upon incubation with the activated C5 and the reacting complement components C6–C9. T-NCl does, however, not cleave any peptide bond in C5 as happens in the physiological activation process but converts the intact protein to the activated form. The conversion is accompanied and probably caused by oxidation of methionine residues in the C5 protein to methionine sulfoxide. Since hypochlorite and T-NCl are biological products generated by the myeloperoxidase-halide system of stimulated leukocytes, the activation of C5 by these agents may be one way to complement activation during inflammation and tissue injury.

Synthetic substrates for coproporophyrinogen oxidase: mesoporphyrinogen-VI revisited

In contradiction to a previous study, mesoporphyrinogen-VI, a dicarboxylate analog of coproporphyrinogen-III, has been shown to be converted to a monovinylic product by chicken red cell hemolysate preparations of coproporphyrinogen oxidase.

Delayed hemolysis of white cell-reduced red cells

TransfusionVolume 34, Issue 1 p. 86-86 Delayed hemolysis of white cell-reduced red cells Tom H. Carson MD, Tom H. Carson MD 1Blood Bank, Children's Hospital 200 Henry Clay Avenue New Orleans. LA 70118Search for more papers by this authorJeannette Bloom MT(ASCP)SBB, Jeannette Bloom MT(ASCP)SBB 1Blood Bank, Children's Hospital 200 Henry Clay Avenue New Orleans. LA 70118Search for more papers by this authorDiane B. Ferguson MT(ASCP)SBB, Diane B. Ferguson MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this authorSusan Thompson MT(ASCP)SBB, Susan Thompson MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this authorBeryl LeBeau MT(ASCP)SBB, Beryl LeBeau MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this author Tom H. Carson MD, Tom H. Carson MD 1Blood Bank, Children's Hospital 200 Henry Clay Avenue New Orleans. LA 70118Search for more papers by this authorJeannette Bloom MT(ASCP)SBB, Jeannette Bloom MT(ASCP)SBB 1Blood Bank, Children's Hospital 200 Henry Clay Avenue New Orleans. LA 70118Search for more papers by this authorDiane B. Ferguson MT(ASCP)SBB, Diane B. Ferguson MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this authorSusan Thompson MT(ASCP)SBB, Susan Thompson MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this authorBeryl LeBeau MT(ASCP)SBB, Beryl LeBeau MT(ASCP)SBB Blood Center for Southeast Louisiana New Orleans, LASearch for more papers by this author First published: January 1994 https://doi.org/10.1046/j.1537-2995.1994.34194098616.xCitations: 5AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume34, Issue1January 1994Pages 86-86 RelatedInformation

Improved separation of acetaldehyde-induced hemoglobin.

A fast-eluting minor variant of hemoglobin A, designated as HbA1-AcH, appears elevated after the incubation of red blood cell hemolysates with acetaldehyde (AcH) and has been proposed as a diagnostic marker for alcoholism or as an indicator for heavy drinking. We have developed an improved HPLC separation of this peak and others elevated by AcH using a polyaspartic acid column (PolyCAT A, PolyLC, Inc.) and a nonlinear buffer gradient with pH changes from 6.6 to 6.8. Saline-washed red blood cells were treated with sodium acetate buffer (pH 5.5) to remove unstable Schiff bases, and then hemolyzed by addition of an equal volume of H2O and 0.4 volumes of CCI4. HbA1-AcH and several others, including two peaks in the HbA1a+b cluster, Hb Pre-A1c, and HbA1d3 were significantly increased by AcH incubation, and the changes were only partially reversible with time. Improved resolution of these peaks allows more accurate quantitation of AcH adducts of hemoglobin.

Antibodies to hydrocortisone in reagent red cells causing positive antibody screening tests

When a patient's serum contains a temperature-independent antibody reacting with all antibody screening and antibody identification panel cells but with few or no units of blood used for crossmatching, one should suspect an antibody against a constituent of the suspension medium of the reagent blood cells. This observation is confirmed by negative reactions with a) washed reagent cells or b) another manufacturer's cells that do not contain the same additives. Two patients are described with antibodies to hydrocortisone that are present in one manufacturer's antibody screening cells and identification panels. The hydrocortisone was added by the manufacturer to prevent hemolysis of the reagent red cells. The patient's antibodies were IgM, complement independent, and nonneutralizable by prior incubation with hydrocortisone. The addition of hydrocortisone to other manufacturers' reagents converted previously negative reactions to the same level of positivity as was seen with the reagent from the index manufacturer. Antibodies to reagent constituents can cause delays in finding compatible blood. It is suggested that manufacturers delete irrelevant additives or those of questionable necessity from their reagents.

Effect of fasting on blood lipid peroxidation parameters of sheep

The effects of three-day fasting and one-day refeeding on some blood metabolites and parameters of lipid peroxidation were studied in eight non-pregnant merino ewes. Fasting produced an immediate decrease in blood glucose accompanied by an increase of free fatty acid, total lipid, total cholesterol and urea in the plasma. Starvation increased the concentration of thiobarbituric acid-reactive substances (malondialdebyde), with a slower but more sustained increase in the plasma than in the red blood cell haemolysate. Changes in glutathione peroxidase activity were the reverse of those in malondialdehyde concentration. Catalase activity was not measurable in plasma but was consistently increased in the haemolysate on fasting. Superoxide dismutase activity in the whole blood haemolysate significantly increased only on the first day of food deprivation. The vitamin E content of plasma showed no significant changes. The results indicate that energy deficiency, a well-known phenomenon in ruminants, affects not only the metabolic parameters of the blood but its lipid peroxidative status as well.

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with 125I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.

Spectrophotometric measurement of plasma 2-thiobarbituric acid-reactive substances in the presence of hemoglobin and bilirubin interference.

The 2-thiobarbituric acid reaction with malondialdehyde has been used to assess lipid peroxidation in a variety of biologic systems. However, in an attempt to measure plasma thiobarbituric acid-reactive substances (TBARS) during extracorporeal membrane oxygenation, a form of sustained cardiopulmonary bypass, it became apparent that the absorbance signal at the 532-nm wavelength was composed not only of the peak absorbance of TBARS, but also of interfering substances from heme pigments and bilirubin. A method of subtracting interfering substances was developed and applied to normal human plasma. The method was tested by adding varying amounts of red blood cell hemolysate, bilirubin, and 1,1,3,3-tetramethoxypropane (TMP) standard to plasma and determining TBARS in the resulting mixture. In addition, varying the amount of added desferoxamine was investigated to determine the effects of iron chelation on the assay. This was important because the different samples would have varying amounts of free iron from hemoglobin to catalyze the reaction. It was found that the following equation could be used in this system to determine that amount of 532-nm absorption due to TBARS: MDA532 = 1.22[(A532) - (0.56)(A510) + (0.44)(A560)]. Regression analysis revealed an 86.6% recovery of the TMP spike. Analysis of variance showed that the variability in the model could be explained mainly by the additive increments of TMP spike (94.6%).

Order of uroporphyrinogen III decarboxylation on incubation of porphobilinogen and uroporphyrinogen III with erythrocyte uroporphyrinogen decarboxylase

The isomeric compositions of the heptacarboxylic, hexacarboxylic and pentacarboxylic porphyrinogens formed by incubation of porphobilinogen with human red-cell haemolysates have been analysed and compared with those derived from incubation with chemically prepared uroporphyrinogen III as substrate. The results indicated that when supplied with an excess (3.7 microM) of exogenous uroporphyrinogen III, uroporphyrinogen decarboxylase utilized the substrate at random and a mixture of isomers was produced; whereas with uroporphyrinogen III generated enzymically from porphobilinogen as substrate a clockwise decarboxylation sequence was observed, resulting in the formation of intermediates mainly with the ring-D, rings-AD and rings-ABD acetate groups decarboxylated. Using [14C]uroporphyrinogen III as substrate at low concentrations (0.01-0.5 microM) also led to preferential decarboxylation of the ring-D acetate group. It was concluded that the order of uroporphyrinogen III decarboxylation is substrate-concentration-dependent, and under normal physiological conditions enzymic decarboxylation is most probably orderly and clockwise, starting at the ring-D acetate group.

A Carboxyfluorescein-Enveloping liposome as a Physicochemical Damage Model of the Biomembrane for the Study of Lipid Peroxidation.

A carboxyfluorescein(CF)-enveloping liposome is presented as a physicochemical damage model of the biomembrane for the study of lipid peroxidation. The degree of membrane damage may be assessed by measurement of the fluorescence intensity of CF released from damaged liposomes after the interaction between the liposomal membrane and a radical initiator. The process of lipid peroxidation was biochemically confirmed by the thiobarbituric acid-reactive substances (TBARS) test done simultaneously. Three types of CF-enveloping liposomes, i.e., soybean phosphatidylcholine (SPC) containing polyunsaturated fatty acid, dipalmitoylphosphatidylcholine (DPPC) containing saturated fatty acid, and soybean phosphatidylcholine plus α-tocopherol, were exposed to a known radical initiator, ferric nitrilotriacetate with a reductant (either cysteine, reduced form glutathione, or ascorbic acid). The release of CF from the peroxidized liposome was measured with a fluorospectrophotometer. A high release of CF and a high level of TBARS were seen in the SPC liposomes, but not in the DPPC and α-tocopherol-inserted SPC liposomes.This liposome model is superior to the conventional red cell hemolysis model for several reasons: (1) the constituents of the membrane are defined; (2) CF is not a radical initiator, whereas hemoglobin is a potential radical initiator; and (3) the membrane components can be easily changed depending on the purpose of the experiment.

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4 + lymphocytes

Functional studies assessed the cytolytic activity of the amino-terminal peptide (FP-I; 23 residues 519–541) of the glycoprotein 41000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37°C, with 40% lysis at 32 μM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526–531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519–524 and residues 526–536) nor a N-terminal flanking peptide (e.g., residues 512–523) induced red cell hemolysis, the entire 23-residue (519–541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4 +-bearing Hut-78 cells, with 40% lysis at approx. 150 μM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327–328).

A rapid method for starting a culture for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines.

We developed a simple and efficient procedure for the establishment of Epstein-Barr virus-transformed human lymphoblastoid cell lines. B-lymphocytes were obtained by centrifugation after hemolysis of red cells with a hemolysis buffer, instead of Ficoll-Parque gradient. We can start a primary culture within 15 min by using this method.

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 μ m free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca + Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 M r), which comprised most (>90%) of the total protein, and an unidentified protein of approximately 13,000 M r. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca + Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 M r (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca + Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 μ m free Ca in a dose-dependent manner over a range of 15 to 100 n m calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 M r protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca + Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller M r protein slightly stimulated the (Ca + Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 μ m free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.