Properties of 3H-cimetidine binding in rat brain membrane fractions

In an attempt to characterize the brain histamine H₂ receptor, experiments were undertaken to study the binding properties of (N-methyl-³H) -cimetidine, an H₂ receptor antagonist, in rat brain membranes. Using a centrifugation assay, ³H-cimetidine binding having a K_d of 0.40μM and a Bmax of 3.9 pmoles/mg protein was detected. Of fourteen anions and cations tested, one, Cu⁺⁺, dramatically increased specific ³H-cimetidine binding, the increase being due mainly to a change in Bmax. Studies of substrate specificity for ³H-cimetidine binding revealed that Cu⁺⁺, while not significantly affecting the potency of H₂ receptor agonists and antagonists, dramatically decreases the potency of H₁ receptor substances on the ³H-cimetidine binding site. In addition, both the relative and absolute potencies of various H₂ receptor agonistsv and antagonists in displacing the ligand in the presence of Cu⁺⁺ parallels their potencies in biological systems. These findings suggest that, under these conditions, ³H-cimetidine may be labelling a biologically relevant H₂ binding site in brain and that Cu⁺⁺ may regulate the substrate specificity for this site. The brain regional distribution and kinetic analysis of the binding suggest that it is not localized solely to the synaptic receptor for histamine, but may also be associated with histamine receptors at other neuronal, glial or vascular sites.     

High affinity 3H-cimetidine binding in guinea-pig tissues

Receptor binding studies have been carried out in guinea-pig cerebral cortex and gastric mucosa membrane preparations using ³H-cimetidine as the radioligand. The binding was found to be time dependent and saturable and confined to a single population of binding sites. However, the calculated K_D values were different for the two tissues, did not correlate with those reported from classical pharmacological experimentation and there was either no or limited displacement by known H₂ specific agonists. It was concluded that the observed high affinity binding site was probably related to an imidazole recognition site rather than the histamine H₂ receptor. The need for careful evaluation of the data is stressed.     

Identification of solubilised beta1 and beta2 adrenoceptors in mammalian lung

The properties of beta-adrenoceptors in solubilised and particulate preparations of rat and rabbit lung have been assessed using the specific ligand (³H)-dihydroalprenolol ((³H)-DHA). Membranes were solubilised using the detergent digitonin and the specific binding of (³H)-DHA assayed using a charcoal-centrifugation technique to separate free and bound ligand. The equilibrium dissociation constant (K_D) of specific (³H)-DHA binding was very similar in particulate and soluble preparations of rat and rabbit lung. Moreover, the optical isomers of propranolol displayed virtually identical stereospecific differences in soluble and membraneous preparations. However, the potency of various catecholamine agonists and the steepness of the displacement curves were greater in all solubilised preparations. Computer-assisted analysis of the displacement curves generated by the highly selective beta₁ antagonist atenolol and the beta₂ antagonist ICI 118.551, revealed the co-presence of beta₁ and beta₂ adrenoceptors in solubilised rabbit lung preparations. Furthermore, soluble beta₁ adrenoceptors appear to be much more labile at 22°C than soluble beta₂ adrenoceptors, providing support for the concept that these receptor subtypes are separate entities.     

Magnesium reduces affinities of antagonists at rat cortex α2-adrenergic receptors labeled with 3H-clonidine: Evidence for heterogeneity of α2-receptor conformations with respect to antagonists

³H-clonidine labeled two binding sites in rat cortex membranes with apparent K_D values of about 1.0 and 5.9 nM. These sites appeared analogous to “super-high” (SH) and “high” (H) affinity states of the α₂-receptor described in human platelets. 10 mM magnesium increased the number of SH receptors by 30% whereas 100 μM GTP reduced SH₃receptor number by 45% with no significant change in the K_D of ³H-clonidine at α₂(SH) sites. In drug competition studies using 1.0 nM ³H-clonidine, 100 μM GTP reduced the affinity of clonidine and increased the affinity of yohimbine, whereas 10 mM magnesium increased the affinity of clonidine and reduced the affinity of yohimbine. The effect of magnesium on the affinity of several antagonists at cortex ³H-clonidine sites ranged from none (phentolamine) to a 6-fold reduction (piperoxan). These data indicate that different states of the α₂-receptor exhibit different affinities for some antagonists.     

Dynamic Measurements of Cerebral Pentose Phosphate Pathway Activity In Vivo Using [1,6-13C2,6,6-2H2] Glucose and Microdialysis

Abstract: Cerebral pentose phosphate pathway (PPP) activity has been linked to NADPH-dependent anabolic pathways, turnover of neurotransmitters, and protection from oxidative stress. Research on this potentially important pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to address this need focused on the use of a novel isotopically substituted glucose molecule, [1,6-¹³C₂,6,6-²H₂]glucose, in conjunction with microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6-¹³C₂,6,6-²H₂]glucose through glycolysis produces [3-¹³C]lactate and [3-¹³C,3,3-²H₂]lactate, whereas metabolism through the PPP produces [3-¹³C,3,3-²H₂]lactate and unlabeled lactate. The ratios of these lactate isotopomers can be quantified using gas chromatography/mass spectrometry (GC/MS) for calculation of PPP activity, which is reported as the percentage of glucose metabolized to lactate that passed through the PPP. Following addition of [1,6-¹³C₂,6,6-²H₂]glucose to the perfusate, labeled lactate was easily detectable in dialysate using GC/MS. Basal forebrain and intracerebral 9L glioma PPP values (mean ± SD) were 3.5 ± 0.4 (n = 4) and 6.2 ± 0.9% (n = 4), respectively. Furthermore, PPP activity could be stimulated in vivo by addition of phenazine methosulfate, an artificial electron acceptor for NADPH, to the perfusion stream. These results show that the activity of the PPP can now be measured dynamically and regionally in the brains of conscious animals in vivo.     

Differential binding of 3H-imipramine and 3H-mianserin in rat cerebral cortex

Drug competition profiles, effect of raphé lesion, and sodium dependency of the binding of two antidepressant drugs ³H-imipramine and ³H-mianserin to rat cerebral cortex homogenate were compared to examine whether the drugs bound to a common “antidepressant receptor.” Of the neurotransmitters tested, only serotonin displaced binding of both ³H-imipramine and ³H-mianserin. ³H-mianserin binding was potently displaced by serotonin S₂ antagonists and exhibited a profile similar to that of ³H-spiperone binding. In the presence of the serotonin S₂ antagonist spiperone, antihistamines (H₁) potently displaced ³H-mianserin binding. ³H-Imipramine binding was displaced potently by serotonin uptake inhibitors. The order of potency of serotonergic drugs in displacing ³H-imipramine binding was not similar to their order in displacing ³H-spiperone or ³H-serotonin binding. Prior midbrain raphé lesions greatly decreased the binding of ³H-imipramine but did not alter binding of ³H-mianserin. Binding of ³H-imipramine but not ³H-mianserin was sodium dependent. These results show that ³H-imipramine and ³H-mianserin bind to different receptors. ³H-Imipramine binds to a presynaptic serotonin receptor which is probably related to a serotonin uptake recognition site, the binding of which is sodium dependent. ³H-Mianserin binds to postsynaptic receptors, possibly both serotonin S₂ and histamine H₁ receptors, the binding of which is sodium independent.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Effect of histamine and H1- and H2-agonists on the fractional outflow of radioactivity from the rat vas deferens preloaded with [3H]-noradrenaline

Histamine and relatively selective H₁- and H₂-receptor agonists caused a concentration-dependent increase in the fractional outflow of radioactivity from the rat isolated vas deferens preloaded with labelled noradrenaline. The evoked fractional outflow of radioactivity was not preferentially associated with either H₁- or H₂-selective agonists, nor was it related to the known potencies of the agonists within each group. The evoked fractional outflow caused by all compounds studied was independent of the extracellular Ca²⁺ concentration. The study cautions against ascribing all the effects of histamine analogs to their respective histamine receptor stimulation.     

New lead elements for histamine H3 receptor ligands in the pyrrolo[2,3-d]pyrimidine class

This work describes the microwave assisted synthesis of twelve novel histamine H₃ receptor ligands. They display pyrrolo[2,3-d]pyrimidine derivatives with rigidized aliphatic amines as warheads. The compounds were screened for H₃R and H₄R binding affinities in radioligand displacement assays and the most potent compounds were evaluated for H₃R binding properties in vitro and in docking studies. The combination of a rigidized H₃R warhead and the pyrrolo[2,3-d]pyrimidine scaffold resulted in selective activity at the H₃ receptor with a pK_i value of 6.90 for the most potent compound. A bipiperidine warhead displayed higher affinity than a piperazine or morpholine motif, while a naphthyl moiety in the arbitrary region increased affinity compared to a phenyl derivative. The compounds can be starting points for novel, simply synthesized histamine H₃ receptor ligands.     

A new approach to the stabilization of dibenzothiophene (DBT) coordination to metals: Studies of (η-C5H4CH2Ph)Ru(CO)2(η(S)-DBT)

One approach to the removal of sulfur-containing compounds from transportation fuels involves the coordination of such compounds to transition metal complexes. We report the design and synthesis of a complex (η⁵-C₅H₄CH₂Ph)Ru(CO)₂(η¹(S)-DBT)⁺ that tests the concept that DBT binding through the sulfur can be augmented by π-π stacking interactions between a pendant phenyl group on the cyclopentadienyl ring and the DBT ligand. The crystal structure and kinetic binding studies of (η⁵-C₅H₄CH₂Ph)Ru(CO)₂(DBT)⁺ are reported and discussed.     

Ontogeny of ppp(A2′p)nA-binding protein in mouse brain

Mouse brain tissue extracts at various stages of development show a drastic change in the specific activity of pp(A2′p)₂A-[³²P]pCp binding protein. Identification of the ppp(A2′p)₃A-[³²P]pCp binding protein was established by (i) binding to the specific ligand ppp(A2′p)₃A-[³²P]pCp, (ii) displacement of binding by nanomolar concentration of pppA(pA)₃, and (iii) affinity labeling techniques in which periodate oxidized ppp(A2′p)₃A-[³²P]pC was specifically cross-linked to a protein with a molecular weight of 86 000. These data suggest that the ppp(A2′p)₃A-[³²P]pCp protein is closely associated with the process of cellular proliferation and differentiation.     

Biochemical Characterization of Two Distinct Angiotensin AT2 Receptor Populations in Murine Neuroblastoma N1E-115 Cells

Abstract: The murine neuroblastoma N1E-115 cell line possesses a high density of angiotensin II (Angll) receptors that can be solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. These solubilized binding sites exhibited high affinity for CGP-42112A and not Losartan, indicating that they were of the AT₂ subtype. However, displacement of ¹²⁵I-Angll with the AT₂ nonpeptide antagonist PD-123319 resulted in a biphasic curve, suggesting heterogeneity of the AT₂ receptor population in N1E-115 cells. In support of this view, separation of two receptor populations was accomplished with heparin-Sepharose chromatography. More specifically, three distinct protein peaks eluted from the heparin-Sepharose column, two of which bound ¹²⁵I-Angll with high affinity and saturability. One of these binding peaks (peak I) eluted rapidly and represented ～80% of the total binding activity, whereas the remaining binding activity was contained within a second peak (peak III) that required the addition of 1.5 M NaCI for its complete elution. Pharmacological analysis revealed that both peaks of binding activity were exclusively AT₂ receptors insofar as they exhibited high affinity for CGP-42112A and little or no affinity for the AT₁-selective antagonist Losartan. However, whereas the nonpeptidic AT₂-selective antagonist PD-123319 completely displaced the binding of ¹²⁶I-Angll from peak I in a monophasic fashion (IC₅₀= 9.1 ± 4.1 nM; mean ± SEM; n = 3), PD-123319 was much less effective in displacing ¹²⁵I-Angll from peak III (IC₅₀= 196 β 27 nM; mean β SEM; n = 3). Treatment of individual peaks with the reducing agent dithiothreitol caused a large increase in ¹²⁵I-Angll specific binding in peak III, whereas a decrease in binding was observed in peak I. Moreover, GTPγS significantly reduced high-affinity agonist binding in peak I but not peak III, further suggesting heterogeneity in the AT₂ receptor family. Finally, immunoblotting studies with polyclonal antisera raised against peak I specifically detected two proteins of 110 and 66 kDa, as is true in crude solubilized membranes, whereas no immunospecific proteins were detected in peak III. These same antisera immunoprecipitated ¹²⁵I-Angll binding activity in peak I but were ineffective in peak III. Collectively, these results suggest that heparin-Sepharose chromatography can efficiently separate two pharmacologically, biochemically and immunologically distinct populations of AT₂ receptors.     

Prostaglandin F2α specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF_2α specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 × 10^?9M and the concentration of binding sites of ～0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF_2α specific binding sites indicates specificity for the 9α-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF_2α analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF_2α binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific ³H-PGE₁ binding could be demonstrated, no high affinity sites could be quantitated. ³H-PGE₁ binding appeared unaffected by changes in temperature or time of incubation, whereas PGF_2α specific binding was significantly modified by both these factors.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Overexpression of Peroxisomal Malate Dehydrogenase MDH3 Gene Enhances Cell Death on H 2O 2 Stress in the ald5 Mutant of Saccharomyces cerevisiae

Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration. With use of the mutant, we examined the detoxication of H₂O₂ generation by fatty acid -oxidation in peroxisome. The ald5 mutant (AKD321), as well as the 746 ⁰ mutant, was more resistant to H₂O₂ stress than the wild type. However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid -oxidation caused a decrease in cell viability of AKD321 to H₂O₂ stress, while the 746 ⁰ mutant had no such effect. Intracellular H₂O₂ concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321. The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321. And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 ⁰ strain, decreasing by 14% to that of 746 ⁰ mutant. The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains. Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress. These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.Received: 23 September 2002 / Accepted: 24 October 2002     

HETEROGENEITY OF HISTAMINE Hi-RECEPTORS: SPECIES VARIATIONS IN [3H]MEPYRAMINE BINDING OF BRAIN MEMBRANES

[³H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H₁receptors. Considerable species differences occur in the affinity of [³H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [³H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H₁ receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [³H]mepyramine binding, exceeding in potency any H₁ antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [³H]mepyramine binding. The regional distribution of specific [³H]mepyramine binding differs considerably in the various species examined.     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.