Enhancing both oral bioavailability and anti-ischemic stroke efficacy of ginkgolide B by preparing nanocrystals self-stabilized Pickering nano-emulsion.

Ginkgolide B (GB), which has been demonstrated as the most efficacious naturally occurring platelet-activating factor (PAF) antagonist, is extensively utilized for the management of cardiovascular and cerebrovascular ailments. Nevertheless, its limited oral bioavailability is hindered by its low solubility in gastric acid and inadequate stability in intestinal fluid, thereby constraining its practical application. This study aimed to develop GB nanocrystals (GB-NCs) and GB nanocrystals self-stabilized Pickering nano-emulsion (GB-NSSPNE) using a miniaturized wet bead milling method. Comparative evaluations were conducted in vivo and in vitro to assess their effectiveness. The findings revealed that GB-NSSPNE, with its intact nanoparticle slow release and absorption, was more effective in enhancing the oral bioavailability of GB compared to the rapid release and absorption of GB-NCs. This finding suggests a potential novel strategy for the oral delivery of GB.

Ultrafast rectifying counter-directional transport of proton and metal ions in metal-organic framework-based nanochannels.

Bioinspired control of ion transport at the subnanoscale has become a major focus in the fields of nanofluidics and membrane separation. It is fundamentally important to achieve rectifying ion-specific transport in artificial ion channels, but it remains a challenge. Here, we report a previously unidentified metal-organic framework nanochannel (MOF NC) nanofluidic system to achieve unidirectional ultrafast counter-directional transport of alkaline metal ions and proton. This highly effective ion-specific rectifying transport behavior is attributed to two distinct mechanisms for metal ions and proton, elucidated by theoretical simulations. Notably, the MOF NC exhibits ultrafast proton conduction stemming from ultrahigh proton mobility, i.e., 11.3 × 10-7 m2 /V·s, and low energy barrier of 0.075 eV in MIL-53-COOH subnanochannels. Furthermore, the MOF NC shows excellent osmotic power-harvesting performance in reverse electrodialysis. This work expects to inspire further research into multifunctional biomimetic ion channels for advanced nanofluidics, biomimetics, and separation applications.

Wafer Bonding of SiC-AlN at Room Temperature for All-SiC Capacitive Pressure Sensor.

Wafer bonding of a silicon carbide (SiC) diaphragm to a patterned SiC substrate coated with aluminum nitride (AlN) film as an insulating layer is a promising choice to fabricate an all-SiC capacitive pressure sensor. To demonstrate the bonding feasibility, a crystalline AlN film with a root-mean-square (RMS) surface roughness less than ~0.70 nm was deposited on a SiC wafer by a pulsed direct current magnetron sputtering method. Room temperature wafer bonding of SiC-AlN by two surface activated bonding (SAB) methods (standard SAB and modified SAB with Si nano-layer sputtering deposition) was studied. Standard SAB failed in the bonding, while the modified SAB achieved the bonding with a bonding energy of ~1.6 J/m2. Both the microstructure and composition of the interface were investigated to understand the bonding mechanisms. Additionally, the surface analyses were employed to confirm the interface investigation. Clear oxidation of the AlN film was found, which is assumed to be the failure reason of direct bonding by standard SAB.

Bidirectional Electrochemiluminescence Color Switch: An Application in Detecting Multimarkers of Prostate Cancer.

A selective excitation of [Ir(df-ppy)2(pic)] and [Ru(bpy)3]2+ through tuning the electrode potential is reported in this work. Bidirectional color change from blue-green to red could be observed along with increase and decrease of the potential, which was ascribed to the dual-potential excitation property of [Ir(df-ppy)2(pic)]. Similar to the three-electrode system, selective excitation of ECL could be achieved at the anode of the bipolar electrode (BPE). Both increase and decrease of the faradic reactions at the cathode of the BPE could induce ECL reporting color at the other pole switched from blue-green to red. We applied a closed BPE device for the bioanalysis of multicolor ECL since the organic solvent containing electrochemiluminophores could be separated from the bioanalytes. On the basis of BPE arrays coupled with the ECL switch, the detection of three biomarkers of prostate cancer, PSA, microRNA-141, and sarcosine were integrated in a same device. The cutoff values of the biomarkers could be recognized directly by the naked eye. Such a device holds great potential in the early diagnosis of prostate cancer.

Antigen-Specific T Cell Analysis Reveals That Active Immune Responses to ß Cell Antigens Are Focused on a Unique Set of Epitopes.

CD38 is an activation marker that is present on recently activated T cells, but absent on resting memory T cells. In this study, we show that CD45RO+CD38+ ß cell Ag-specific CD4+ T cells were present at higher frequencies in type 1 diabetes subjects compared with those in healthy subjects. These results imply an ongoing ß cell immunity years after onset of diabetes and suggest these activated T cells have an active role in the disease process. The Ag specificities of these activated T cells were determined by a novel CD154 T cell epitope mapping assay. Although each patient usually had a unique set of epitopes recognized by these T cells, two epitopes, DR0401-restricted modified preproinsulin peptide 78-90K88S and zinc transport 8 266-285, were repeatedly identified in multiple subjects. Identifying these T cells and their specific antigenic epitopes might provide immunotherapeutic targets for personalized therapies.

Genetically Encoded Azide Containing Amino Acid in Mammalian Cells Enables Site-Specific Antibody-Drug Conjugates Using Click Cycloaddition Chemistry.

Antibody-drug conjugates (ADC) have emerged as potent antitumor drugs that provide increased efficacy, specificity, and tolerability over chemotherapy for the treatment of cancer. ADCs generated by targeting cysteines and lysines on the antibody have shown efficacy, but these products are heterogeneous, and instability may limit their dosing. Here, a novel technology is described that enables site-specific conjugation of toxins to antibodies using chemistry to produce homogeneous, potent, and highly stable conjugates. We have developed a cell-based mammalian expression system capable of site-specific integration of a non-natural amino acid containing an azide moiety. The azide group enables click cycloaddition chemistry that generates a stable heterocyclic triazole linkage. Antibodies to Her2/neu were expressed to contain N6-((2-azidoethoxy)carbonyl)-l-lysine at four different positions. Each site allowed over 95% conjugation efficacy with the toxins auristatin F or a pyrrolobenzodiazepine (PBD) dimer to generate ADCs with a drug to antibody ratio of >1.9. The ADCs were potent and specific in in vitro cytotoxicity assays. An anti Her2/neu conjugate demonstrated stability in vivo and a PBD containing ADC showed potent efficacy in a mouse tumor xenograph model. This technology was extended to generate fully functional ADCs with four toxins per antibody. The high stability of the azide-alkyne linkage, combined with the site-specific nature of the expression system, provides a means for the generation of ADCs with optimized pharmacokinetic, biological, and biophysical properties.

AZ17: a new bispecific drug targeting IL-6 and IL-23 with potential clinical use--improves psoriasis in a human xenograft transplantation model.

Targeting more than one molecule in multifactorial diseases involving several disease mediators may provide improved therapeutic efficacy. Psoriasis is a multifactorial disease in which interleukin (IL)-6 and IL-23 are important disease mediators because they facilitate development of Th17 cells; widely accepted to be associated with psoriasis. To meet the need for new therapeutics, we aimed to create a clinically relevant bispecific drug, by combining the inhibitory properties of anti-IL-6 and anti-IL-23 antibodies, exhibiting high affinity, high stability and the ability to be produced in high yield. The bispecific molecule AZ17 was created by combining high affinity binding domains originating from monoclonal antibodies targeting human IL-6 and IL-23. To allow for high and efficient production, AZ17 was assembled by site-specific bioconjugation from two individual single chain fragment variables that were synthesized separately in Escherichia coli. To improve stability and extend pharmacokinetics, a flexible poly-ethylene glycol molecule was used as linker. In preclinical psoriasis models, AZ17 reduced IL-23-induced ear inflammation and improved psoriasis in a xenograft transplantation model where psoriasis skin is transplanted onto immune-deficient mice. The data presented here suggest AZ17 to be a promising drug candidate in psoriasis and other inflammatory diseases associated with Th17 cell development.

The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization.

The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9). However, changes at position 675 in conjunction with Thr to Ala at position 569 increased the neutralization sensitivity to all gp41 and gp120 MAbs and plasma, in some cases by more than 1000-fold. Interestingly, the T569A change had a dramatic effect on b12 binding, but no effect on neutralization sensitivity. This finding suggests that antibody neutralization may occur through a multi-step pathway that includes distinct changes in envelope conformation that may affect binding but not neutralization susceptibility.

Binding interactions between soluble HIV envelope glycoproteins and quaternary-structure-specific monoclonal antibodies PG9 and PG16.

PG9 and PG16 are antibodies isolated from a subject infected with HIV-1 and display broad anti-HIV neutralizing activities. They recognize overlapping epitopes, which are preferentially expressed on the membrane-anchored trimeric form of the HIV envelope glycoprotein (Env). PG9 and PG16 were reported not to bind to soluble mimetics of Env. The engineering of soluble Env proteins on which the PG9 and PG16 epitopes are optimally exposed will support efforts to elicit broad anti-HIV neutralizing antibodies by immunization. Here, we identified several soluble gp140 Env proteins that are recognized by PG9 and PG16, and we investigated the molecular details of those binding interactions. The IgG versions of PG9 and PG16 recognize the soluble trimeric gp140 form less efficiently than the corresponding monomeric gp140 form. In contrast, the Fab versions of PG9 and PG16 recognized the monomeric and trimeric gp140 forms with identical binding kinetics and with binding affinities similar to the high binding affinity of the anti-V3 antibody 447D to its epitope. Our data also indicate that, depending on the Env backbone, the interactions of PG9 and PG16 with gp140 may be facilitated by the presence of the gp41 ectodomain and are independent of the proper enzymatic cleavage of gp140 into gp120 and gp41. The identification of soluble Env proteins that express the PG9 and PG16 epitopes and the detailed characterization of the molecular interactions between these two antibodies and their ligands provide important and novel information that will assist in improving the engineering of future Env immunogens.

Computational protein design using flexible backbone remodeling and resurfacing: case studies in structure-based antigen design.

Computational protein design has promise for vaccine design and other applications. We previously transplanted the HIV 4E10 epitope onto non-HIV protein scaffolds for structural stabilization and immune presentation. Here, we developed two methods to optimize the structure of an antigen, flexible backbone remodeling and resurfacing, and we applied these methods to a 4E10 scaffold. In flexible-backbone remodeling, an existing backbone segment is replaced by a de novo designed segment of prespecified length and secondary structure. With remodeling, we replaced a potentially immunodominant domain on the scaffold with a helix-loop segment that made intimate contact to the protein core. All three domain trim designs tested experimentally had improved thermal stability and similar binding affinity for the 4E10 antibody compared to the parent scaffold. A crystal structure of one design had a 0.8 Å backbone RMSD to the computational model in the rebuilt region. Comparison of parent and trimmed scaffold reactivity to anti-parent sera confirmed the deletion of an immunodominant domain. In resurfacing, the surface of an antigen outside a target epitope is redesigned to obtain variants that maintain only the target epitope. Resurfaced variants of two scaffolds were designed in which 50 positions amounting to 40% of the protein sequences were mutated. Surface-patch analyses indicated that most potential antibody footprints outside the 4E10 epitope were altered. The resurfaced variants maintained thermal stability and binding affinity. These results indicate that flexible-backbone remodeling and resurfacing are useful tools for antigen optimization and protein engineering generally.

Computational design of epitope-scaffolds allows induction of antibodies specific for a poorly immunogenic HIV vaccine epitope.

Broadly cross-reactive monoclonal antibodies define epitopes for vaccine development against HIV and other highly mutable viruses. Crystal structures are available for several such antibody-epitope complexes, but methods are needed to translate that structural information into immunogens that re-elicit similar antibodies. We describe a general computational method to design epitope-scaffolds in which contiguous structural epitopes are transplanted to scaffold proteins for conformational stabilization and immune presentation. Epitope-scaffolds designed for the poorly immunogenic but conserved HIV epitope 4E10 exhibited high epitope structural mimicry, bound with higher affinities to monoclonal antibody (mAb) 4E10 than the cognate peptide, and inhibited HIV neutralization by HIV+ sera. Rabbit immunization with an epitope-scaffold induced antibodies with structural specificity highly similar to mAb 4E10, an important advance toward elicitation of neutralizing activity. The results demonstrate that computationally designed epitope-scaffolds are valuable as structure-specific serological reagents and as immunogens to elicit antibodies with predetermined structural specificity.

The structural basis of 5' triphosphate double-stranded RNA recognition by RIG-I C-terminal domain.

RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5' triphosphate (ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here, we show that RIG-I CTD binds 5' ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5' ppp dsRNA. Crystal structures of RIG-I CTD bound to 5' ppp dsRNA with GC- and AU-rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5' ppp through extensive electrostatic interactions. Mutagenesis and RNA-binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA-binding surface affected RIG-I signaling in cells.

Interactions between lipids and human anti-HIV antibody 4E10 can be reduced without ablating neutralizing activity.

Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV infections, through elimination by B-cell tolerance mechanisms to self-antigens, or to contribute to neutralization potency by virus-specific membrane binding outside of the membrane-proximal external region (MPER). To investigate how 4E10 interacts with membrane and protein components, and whether such interactions contribute to neutralization mechanisms, we introduced two mutations into 4E10 Fv constructs, Trp to Ala at position 100 in the heavy chain [W(H100)A] and Gly to Glu at position 50 in the light chain [G(L50)E], selected to disrupt potential lipid interactions via different mechanisms. Wild-type and mutant Fvs all bound with the same affinity to peptides and monomeric and trimeric gp140s, but the affinities for gp140s were uniformly 10-fold weaker than to peptides. 4E10 Fv binding responses to liposomes in the presence or absence of MPER peptides were weak in absolute terms, consistent with prior observations, and both mutations attenuated interactions even further, as predicted. The W(H100)A mutation reduced neutralization efficiency against four HIV-1 isolates, but the G(L50)E mutation increased potency across the same panel. Electron paramagnetic resonance experiments showed that the W(H100)A mutation, but not the G(L50)E mutation, reduced the ability of 4E10 to extract MPER peptides from membranes. These results show that 4E10 nonspecific membrane binding is separable from neutralization, which is achieved through specific peptide/lipid orientation changes.

Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5.

RIG-I, MDA5 and LGP2 are cytosolic pattern recognition receptors detecting single-stranded or double-stranded RNA in virally infected cells. The activation of RIG-I or MDA5 stimulates the secretion of type I interferons that play key roles in antiviral immune responses. The C-terminal domains (CTD) of RIG-I and LGP2 are responsible for RNA binding; however, it is not clear how MDA5 binds RNA. To understand the structural basis of dsRNA recognition by MDA5, we have determined the 1.45A resolution structure of the C-terminal domain of human MDA5. The structure revealed a highly conserved fold similar to the structures of RIG-I and LGP2 CTDs. NMR titration of MDA5 CTD with dsRNA demonstrated that a positively charged surface is involved in dsRNA binding. Mutagenesis and RNA binding studies showed that electrostatic interactions play primary roles in dsRNA recognition by MDA5. Like RIG-I and LGP2, MDA5 CTD preferentially binds dsRNA with blunt ends, but does not associate with dsRNA with either 5' or 3' overhangs. Molecular modeling of MDA5 CTD/dsRNA complex suggests that MDA5 CTD may recognize the first turn of blunt-ended dsRNA in a similar manner as LGP2.

Crystal structures of ligand-bound saccharopine dehydrogenase from Saccharomyces cerevisiae.

Three structures of saccharopine dehydrogenase (l-lysine-forming) (SDH) have been determined in the presence of sulfate, adenosine monophosphate (AMP), and oxalylglycine (OxGly). In the sulfate-bound structure, a sulfate ion binds in a cleft between the two domains of SDH, occupies one of the substrate carboxylate binding sites, and results in partial closure of the active site of the enzyme due to a domain rotation of almost 12 degrees in comparison to the apoenzyme structure. In the second structure, AMP binds to the active site in an area where the NAD+ cofactor is expected to bind. All of the AMP moieties (adenine ring, ribose, and phosphate) interact with specific residues of the enzyme. In the OxGly-bound structure, carboxylates of OxGly interact with arginine residues representative of the manner in which substrate (alpha-ketoglutarate and saccharopine) may bind. The alpha-keto group of OxGly interacts with Lys77 and His96, which are candidates for acid-base catalysis. Analysis of ligand-enzyme interactions, comparative structural analysis, corroboration with kinetic data, and discussion of a ternary complex model are presented in this study.

Determinants of substrate specificity for saccharopine dehydrogenase from Saccharomyces cerevisiae.

A survey of NADH, alpha-Kg, and lysine analogues has been undertaken in an attempt to define the substrate specificity of saccharopine dehydrogenase and to identify functional groups on all substrates and dinucleotides important for substrate binding. A number of NAD analogues, including NADP, 3-acetylpyridine adenine dinucleotide (3-APAD), 3-pyridinealdehyde adenine dinucleotide (3-PAAD), and thionicotinamide adenine dinucleotide (thio-NAD), can serve as a substrate in the oxidative deamination reaction, as can a number of alpha-keto analogues, including glyoxylate, pyruvate, alpha-ketobutyrate, alpha-ketovalerate, alpha-ketomalonate, and alpha-ketoadipate. Inhibition studies using nucleotide analogues suggest that the majority of the binding energy of the dinucleotides comes from the AMP portion and that distinctly different conformations are generated upon binding of the oxidized and reduced dinucleotides. Addition of the 2'-phosphate as in NADPH causes poor binding of subsequent substrates but has little effect on coenzyme binding and catalysis. In addition, the 10-fold decrease in affinity of 3-APAD in comparison to NAD suggests that the nicotinamide ring binding pocket is hydrophilic. Extensive inhibition studies using aliphatic and aromatic keto acid analogues have been carried out to gain insight into the keto acid binding pocket. Data suggest that a side chain with three carbons (from the alpha-keto group up to and including the side chain carboxylate) is optimal. In addition, the distance between the C1-C2 unit and the C5 carboxylate of the alpha-keto acid is also important for binding; the alpha-oxo group contributes a factor of 10 to affinity. The keto acid binding pocket is relatively large and flexible and can accommodate the bulky aromatic ring of a pyridine dicarboxylic acid and a negative charge at the C3 but not the C4 position. However, the amino acid binding site is hydrophobic, and the optimal length of the hydrophobic portion of the amino acid carbon side chain is three or four carbons. In addition, the amino acid binding pocket can accommodate a branch at the gamma-carbon, but not at the beta-carbon.

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae.

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.

The alpha-aminoadipate pathway for lysine biosynthesis in fungi.

This review provides a description of the biochemistry and enzymology of the alpha-aminoadipate pathway for lysine biosynthesis in fungi. The alpha-aminoadipate pathway is unique to fungi and is thus a potential target for the rational design of antifungal drugs. The present state of knowledge of the mechanisms of the seven enzymes in the pathway is presented, as well as detailed information with respect to structures and mechanisms of homocitrate synthase, saccharopine reductase, and saccharopine dehydrogenase.

Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae.

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.