Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats

Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.     

Effect of hemoglobin A and S on human erythrocyte ghosts

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.     

Ca2+- and Mg-ATP-dependent shape change of human erythrocyte ghosts and triton shells

Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+.     

Solubility of palmitoyl-coenzyme A in acyltransferase assay buffers containing magnesium ions

The solubility of palmitoyl-CoA is strongly affected by Mg2+ concentrations commonly used in acyltransferase reactions. In 0.10 M Tris-HCl buffer at pH 7.4 or 8.5, all of the palmitoyl-CoA in 10 microM solutions and 90% of the palmitoyl-CoA in 100 microM solutions are precipitated by 1 mM Mg2+. In 0.05 M phosphate at pH 7.4, and in 0.10 M Tris-HCl containing 0.4 M KCl, the substrate remains soluble at Mg2+ concentrations below 4-5 mM. Above 5 mM Mg2+, palmitoyl-CoA is insoluble in all of these buffers. Substrate solubility could therefore be a limiting factor when free Mg2+ and fatty acyl-CoAs are present together during acyltransferase assays.     

The labeling with 8-azido-cyclic adenosine monophosphate of proteins in vesicles of sarcoplasmic reticulum from rabbit skeletal muscle

The technique of laser Doppler electrophoresis was applied for the study of the surface charge properties of (NA+, K+)-ATPase containing microsomal vesicles derived from guinea-pig kidney. The influence of pH, the screening and binding of uni- and divalent cations and the binding of ATP show: (1) one net negative charge per protein unit with a pK = 3.9; (2) deviation from the Debye relation between surface potential and ionic strength for univalent cations, with no difference in the effect of Na+ and K+; (3) Mg2+ binds with an association constant of Ka = 1.1. 10(2) M-1 while ATP binds with an apparent Ka = 1.1.10(4) M-1 for 1 mM NaCl, 0.2 mM KCI, 0.1 mM MgCl2, 0.1 mM Tris-HCl2, 0.1 mM Tris-HCl (pH 7.3). The binding is weaker at higher Mg2+ concentrations. There is no ATP binding in the absence of Mg2+. In addition, the average vesicle size derived from the linewidth of the quasielastic light scattering spectrum is 203.7 +/- 15.2 nm. In the presence of ATP a reduction in size is observed.     

Na+-like effect of imidazole on the phosphorylation of (Na+ + K+)-ATPase

A high basal level of phosphorylation (approx. 70% of the optimal Na+-dependent phosphorylation level) is observed in 50 mM imidazole-HCl (pH 7.0), in the absence of added Na+ and K+ and the presence of 10-100 microM Mg2+. In 50 mM Tris-HCl (pH 7.0) the basal level is only 5%, irrespective of the Mg2+ concentration. Nevertheless, imidazole is a less effective activator of phosphorylation than Na+ (Km imidazole-H+ 5.9 mM, Km Na+ 2 mM under comparable conditions). Imidazole-activated phosphorylation is strongly pH dependent, being optimal at pH less than or equal to 7 and minimal at pH greater than or equal to 8, while Na+-activated phosphorylation is optimal at pH 7.4. This suggests that imidazole-H+ is the activating species. Imidazole facilitates Na+-stimulated phosphorylation. The Km for Na+ decreases from 0.63 mM at 5 mM imidazole-HCl to 0.21 mM at 50 mM imidazole-HCl (pH 7; 0.1 mM Mg2+ in all cases). Imidazole-activated phosphorylation is more sensitive to inhibition by K+ (I50 = 12.5 microM) than Na+-activated phosphorylation (I50 = 180 microM). Mg2+ antagonizes activation by imidazole-H+ and also inhibition by K+. The Ki value for Mg2+ (approx. 0.3 mM) is the same for the two antagonistic effects. Tris buffer (pH 7.0) inhibits imidazole-activated phosphorylation with an I50 value of 30 mM in 50 mM imidazole-HCl (pH 7.0) plus 0.1 mM Mg2+. We conclude that imidazole-H+, but not Tris-H+, can replace Na+ as an activator of ATP-dependent phosphorylation, primarily by shifting the E2----E1 transition to the right, leading to a phosphorylating E1 conformation which is different from that in Tris buffer.     

Separation of the lipid bilayer from the membrane skeleton during discocyte-echinocyte transformation of human erythrocyte ghosts

The membrane skeleton, a protein lattice at the internal side of the red cell membrane, is principally composed of spectrin, actin and proteins 4.1 and 4.9. We have examined negatively stained red cell ghosts and demonstrated, on an ultrastructural level, a separation of the lipid bilayer from the membrane skeleton during echinocytic transformation. The electron micrographs of discoidal red cell ghosts suspended in hypotonic buffer revealed a filamentous reticulum that uniformly laminated the entire submembrane region. transformation of the discoidal ghosts into echinocytic form, as induced by incubation in isotonic buffer, resulted in a disruption of skeletal continuity underlying the surface contour of the membrane spicule. The submembrane reticulum extended into the base and the neck of the spiny processes of the crenated ghosts but was absent at the tip of these projections. In addition, membrane vesicles without a submembrane reticulum were detected either attached to the tips of the spicules or released into the supernatant from the echinocytic ghosts. Protein analysis revealed that the released vesicles were enriched in bands 3, 4.1 and 7 and contained very little of the membrane skeletal proteins, spectrin and actin. The data indicate that during echinocyte formation, parts of the lipid bilayer physically separate from the membrane skeleton, leading to a formation of skeleton-poor lipid vesicles.     

Passive Ca2+ fluxes across the membrane of sarcoplasmatic reticulum of skeletal muscles. The effect of calcium channel blockers

It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes.     

Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase.

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35–40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C-and N-terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

Isolation of the protein B23/nucleophosmin from HeLa cell nuclei

Endogenous forms of the protein B23 were for the first time isolated from HeLa cell nuclei and their structural states were analyzed. It was demonstrated that incubation of HeLa cell nuclei in 10 mM Tris-HCl buffer (pH 7.4) led, not only to their swelling, but also to the release of several nuclear proteins, including the protein B23. PAGE of the supernatant fraction allowed nine major stained protein bands to be detected; the bands were identified by MALDI mass spectrometry (matrix-assisted laser desorption and ionization). The proteins in the range of 35-40 kDa were identified as nucleophosmin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1. Analysis of the N- and C-terminal amino acid sequences showed the presence of the isoforms B23.1 and B23.2, GAPDH, and the isoform hnRNP B1 and made it possible to describe the C- and N- terminal processing patterns and demonstrate the presence of isoform B23.2 at a protein level.     

Mechanism of Cu2+-induced elevation of [3H]cimetidine binding to membranes in rat brain

The mechanism of increasing effect of CuCl₂ on specific [³H]cimetidine binding was examined in brain membranes of rats. CuCl₂-Induced elevation of [³H]cimetidine binding was high in Krebs-Ringer solution (pH 7.4) compared to those in 50 mM Na, K-phosphate buffer (pH 7.4) and in 50 mM Tris-HCl buffer (pH 7.4). CaCl₂ (5–50 mM) inhibited effect of CuCl₂, but NaCl (25–200 mM), KCl (5–100 mM) or MgCl₂ (5–50 mM) did not. CuCl₂ (50 μM) elevated 9.3- and 2.5-fold the binding in phosphate- and Tris—HCl buffer, respectively. EDTA-2Na decreased the binding elevated by 50 μM CuCl₂ in phosphate buffer to the similar level in Tris-HCl buffer, whereas it did not affect those in Tris-HCl buffer. The absorption spectra of cimetidine and CuCl₂ mixture showed a peak at 317 nm in phosphate buffer that was not observed in Tris-HCl buffer. It is suggested that cimetidine-Cu²⁺ chelate complex could be formed in phosphate buffer, resulting in higher amount of binding in phosphate buffer than in Tris-HCl buffer. PdCl₂ also caused a marked elevation in [³H]cimetidine binding, seeming to be due to formation of cimetidine-Pd²⁺ chelate complex. There were two types of [³H]cimetidine binding in the presence of 20 nM PdCl₂: high affinity binding with K_d = 0.7 ± 0.1 nM and low affinity binding with K_d = 44.3 ± 3.0 nM. It is suggested that cimetidine-Cu²⁺ complex binds to cimetidine binding sites in brain with higher affinity than cimetidine alone.     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Characteristics of ceruloplasmin interaction with a specific receptor of human erythrocytes

The equilibrium binding of ([125I]ceruloplasmin) ([125I]CP) to a specific receptor of human erythrocytes was investigated. It was shown that reaching the binding equilibrium is a slow process. A strong dependence of binding on Ca2+ concentration (from 0.1 to 1 mM) was revealed; the optimal values were achieved at millimolar concentrations of Ca2+.Mg2+ do not affect the binding of [125I]CP. Under conditions of optimal binding (0.01 M Tris-HCl buffer pH 7.4 containing 158 mM NaCl and 1 mM Ca2+, 4 degrees C), the values of constants for [125I]CP binding to intact erythrocytes (Kd = 1.0 nm) and to membrane fragments (Kd = 0.8 nM) as well as the number of binding sites (16.3 X 10(-15) mol per 40,000,000 erythrocytes) were determined. No ceruloplasmin transport across the erythrocyte membrane was observed. This finding and the similarity of Kd values for ceruloplasmin binding to membrane fragments and to intact erythrocytes indicate that the effect of ceruloplasmin on human erythrocytes is due to the protein molecule interaction with membrane receptors.     

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes

The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Factors of the shape change of human erythrocytes induced with lidocaine

We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lidocaine when ATP was present. But, the shape of resealed cells which were prepared with 10 mM Tris-HCl buffer (pH 7.4) containing 2 mM ATP-MgCl2 and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HCl buffer (pH 7.4) were incubated with various concentrations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These membranous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.