A method for preparing analytically pure sodium dithionite. Dithionite quality and observed nitrogenase-specific activities.

Sodium dithionite (Na2S2O4) is widely used as a reductant in biochemical studies, but has not been available in its pure form. A convenient, detailed procedure is given for the recrystallization of commercial dithionite from 0.1 M NaOH-methanol under anaerobic conditions. Twice-recrystallized dithionite had a purity of 99 +/- 1% by UV spectroscopy (A315) and elemental analysis. The influence of dithionite quality on the apparent reduction activities of the nitrogenase components (Av1 and Av2) from Azotobacter vinelandii was investigated.

Thermodynamic model of cooperativity in a dimeric protein: unique and independent parameters formulation.

A model of the cooperative interaction of ligand binding to a dimeric protein is presented based upon the unique and independent parameters (UIP) thermodynamic formulation (Gutheil and McKenna, Biophys. Chem. 45 (1992) 171-179). The analysis is developed from an initial model which includes coupled conformational and ligand binding equilibria. This completely general model is then restricted to focus on conformationally mediated cooperative interactions between the ligands and the expressions for the apparent ligand binding constant and the apparent ligand-ligand interaction constant are derived. The conditions under which there is no cooperative interaction between the ligands are found as roots to a polynomial equation. Consideration of the distribution of species among the various conformational states in this general model leads to a set of inequalities which can be represented as a two dimensional plot of boundaries. By superimposing a contour plot of the value of the apparent ligand-ligand interaction constant over the plot of boundaries a complete graphical representation of this system is achieved similar to a phase diagram. It is found that the parameter space homologous to Koshland-Nemethy-Filmer type of model is most consistent with both positive and negative cooperativity in this model. The maximal amount of positive and negative cooperativity are found to be simple functions of Kc, the equilibrium constant associated with the change of a subunit and ligand from the unligated to ligated conformation. It is shown that under certain limiting conditions the apparent allosteric interaction between ligands is equal to the conformational interaction between subunits. The methods presented are generally applicable to the theoretical analysis of thermodynamic interactions in complex systems.

Reformulation of thermodynamic systems with aggregation and theoretical methods for the analysis of ligand binding in proteins with monomer-multimer equilibria.

The reformulation of complex thermodynamic systems is a useful tool for their analysis as demonstrated by the theoretical analysis of conformationally mediated cooperativity in a dimeric protein. Many chemical and biochemical systems exhibit monomer-multimer equilibria, behavior not addressed in the original reformulation. A method for reformulating such systems, and the mathematical methods necessary for relating alternative models, are therefore developed. The basic principles of the reformulation are illustrated on homodimeric and heterodimeric systems. The mathematical methods necessary to relate alternative models are then derived from probabilistic considerations. Higher-order models (more interacting subunits) are related to lower-order models (fewer interacting subunits) by a polynomial expansion of the sum of species in the lower-order model to give the sum of species in the higher-order model. Using these methods, the equations describing the ligand binding behavior of a homomeric monomer-dimer system are derived. These methods are also used to relate the two alternative models for cooperativity for a homotetrameric protein; one model where the dimer is the cooperative unit and the other where the tetramer is the cooperative unit.

Application of hierarchical thermodynamic interactions to the protonation equilibria of organic polyprotic acids.

A general method for formulating complex thermodynamic systems in terms of hierarchical interactions has been developed, and has been applied in a previous analyses to the theoretical analysis of cooperativity in a dimeric protein, to the statistical analysis of hemoglobin oxygen binding data, and to the protonation equilibria of inorganic polyprotic acids. Organic polyprotic acids have served as a demonstration system for the development of concepts and methods for treating complex biochemical equilibria. Glutamic acid is the classic test case for understanding proton-proton interactions in organic polyprotic acids, and this system is analyzed using the concept of hierarchical interactions. Second order interactions were apparent between all three possible proton interactions, as has been established previously. The third order interaction between the three protons was found to be insignificant, indicating that protonation of one site on glutamate has no effect on the interaction between the other two protonation sites. This further reinforces the premise that higher order terms, representing more complex interactions, are less likely to be significant than lower order terms. To allow correlation of the interaction values from glutamate with other organic acids, pairwise interaction values between protonation events were then calculated from known pKd values for a number of diprotic acids and bases. For simple straight chain acids and bases a linear log-log relationship was apparent between the number of intervening atoms between the protons and the pK(d,hh) (pKd of interaction). This relationship extended from three atoms (carbonate) up to 11 atoms (azelaic acid) and applied to both dicarboxylic acids and diamine bases. The pairwise interactions in glutamate also followed this simple relationship.

A simple chemical example of hierarchical thermodynamic interactions: the protonation equilibria of inorganic polyprotic acids.

A general method for formulating complex thermodynamic systems in terms of hierarchical interactions has been developed, and has been applied in a previous analysis to hemoglobin oxygen binding data. Polyprotic acids can be considered a simple chemical model of thermodynamic interaction between ligand binding events. To further illustrate the hierarchical interaction approach it is applied to the analysis of the thermodynamic interactions between proton binding events in inorganic polyprotic acids. pK values for arsenate, carbonate, chromate, phosphate, phosphite, selenite, sulfide and sulfite were recast into hierarchical interaction terms. The intrinsic K(d,h) for protonation ranged from 8.8 x 10(-13) (M) for phosphate to 1.3 x 10(-6) (M) for chromate. Pairwise interactions (K(d,hh)) between protonation events ranged from 1.3 x 10(4) for phosphite to 9.4 x 10(5) for carbonate. Third order interactions (K(d,hhh)) were 0.91 and 0.51 for arsenate and phosphate, respectively, values relatively close to the no interaction value of 1. A principle feature of systems described by hierarchical interactions is that higher order interactions, representing more complex interactions, are less likely to be significant than lower order interactions, and this is further illustrated by these observations from polyprotic acids. The set of significant hierarchical interaction values can be used to predict values for as yet unobserved events, and projected pK values are made for all the polyprotic acids included in this study. Finally, application of this method to the protonation equilibria of water demonstrates a profound pairwise interaction between protonation events (K(d,hh) = 1.3 x 10(17)), which is attributed to oxygen's small size and lack of polarizability.

Statistical analysis of data pertaining to complex state systems by stepwise regression with reformulated parameters; application to spectroscopically monitored hemoglobin oxygen binding data.

A method is described for the statistical analysis of data pertaining to complex state systems, based on the concept of reformulating the parameters describing the system as a hierarchy of interactions, and this method demonstrated on the analysis of spectroscopically monitored hemoglobin oxygen binding data [K. Imai, Biophys. Chem. 37 (1990) 197-210]. The concept of reformulation was first extended to state parameters other than delta G degree s, such as the extinction coefficients (epsilon s) associated with different ligation states during hemoglobin oxygen binding. The reformulated parameters are incrementally allowed to vary in the data fitting procedure, and the statistical significance of the added parameters tested by F and Kolmogorov-Smirnov tests. The result of this method is the minimal set of statistically significant parameters required to describe the data. The hierarchical nature of reformulated parameters allows the physical significance of the subset of statistically significant parameters to be discussed even when all reformulated terms may not be statistically significant. Applying this method to hemoglobin oxygen binding data with the reformulated Adair model demonstrated that at least two, and at most three, of the four reformulated Adair constants are statistically significant. A reformulated square model was found to give a statistically indistinguishable fit from the Adair model, with the statistically significant thermodynamic terms essentially those proposed by Linus Pauling in 1935. A change in delta epsilon with subsequent oxygen binding events was found to be significant in both models. These results are consistent with a model for hemoglobin oxygen binding where a subunit changes its conformation upon oxygen binding, and affects the conformation of adjacent subunits.

Unique and independent parameters (UIP) formulation for thermodynamic models of complex protein-ligand systems.

A method for reformulating the thermodynamic (delta G) description of complex equilibria is presented. The purpose of this reformulation is to take a system of N complexes which is completely defined by N delta Gs, and recast it in terms of a new set of N delta Gs. This reformulation is an extension of the concept of interaction energy (J. Wyman, Adv. Protein Chem. 19 (1964) 223-286). The new delta Gs obtained by this reformulation reflect the intrinsic properties of the binding sites and the hierarchical nature of potential interactions between them. A simple set of rules are developed which allow for the description of complex protein-ligand binding schemes and these rules are used to derive schemes for hemoglobin O2 binding. This reformulation represents the foundation for the theoretical description of the coupling of energy in protein-ligand systems as illustrated by the theoretical analysis of allosterism in a dimeric protein presented in the following paper. This reformulation also provides the foundation for the analysis of data pertaining to complex equilibria.

A sensitive equilibrium-based assay for D-lactate using D-lactate dehydrogenase: application to penicillin-binding protein/DD-carboxypeptidase activity assays.

An assay for D-lactate (D-Lac) is described where D-Lac in the presence of NAD+ is equilibrated to NADH and pyruvate (Pyr) by Leuconostoc mesenteroides D-lactate dehydrogenase (DLDH). This assay was standardized using known concentrations of D-Lac and a linearized form of the equilibrium expression. The assay has a lower limit of about 20 microM D-Lac in a 1 ml assay mixture (20 nmol D-Lac). As a demonstration of this assay method it is used to characterize the hydrolysis of the standard penicillin-binding protein/DD-carboxypeptidase substrate Ac2-L-Lys-D-Ala-D-Lac by penicillin-binding protein 5 from Escherichia coli. The approach adopted here of using an inherently nonlinear response, which however follows precisely determinable physical behavior, has the advantages of providing a wider dynamic range and increased relative precision over analogous linear response-based methods. This approach may be applicable to the development of other enzyme-based assay methods.

Separation of L-Pro-DL-boroPro into its component diastereomers and kinetic analysis of their inhibition of dipeptidyl peptidase IV. A new method for the analysis of slow, tight-binding inhibition.

The potent dipeptidyl peptidase IV (DP IV) inhibitor [1-(2-pyrrolidinylcarbonyl)-2-pyrrolidinyl]boronic acid (L-Pro-DL-boroPro) [Flentke, G. R., Munoz, E., Huber, B. T., Plaut, A. G., Kettner, C. A., & Bachovchin, W. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1556-1559] was fractionated into its component L-L and L-D diastereomers by C18 HPLC, and the binding of the purified diastereomers to DP IV was analyzed. Inhibition kinetics confirms that the L-L diastereomer is a potent inhibitor of DP IV, having a Ki of 16 pM. The L-D isomer binds at least 1000-fold more weakly than the L-L, if it binds at all, as the approximately 200-fold weaker inhibition observed for the purified L-D isomer is shown here to be due entirely to the presence of a small amount (0.59%) of the L-L diastereomer contaminating the L-D preparation. The instability of Pro-boroPro, together with its very high affinity for DP IV and the time dependence of the inhibition, makes a rigorous kinetic analysis of its binding to DP IV difficult. Here we have developed a method which takes advantage of the slow rate at which the inhibitor dissociates from the enzyme. The method involves preincubating the enzyme and the inhibitor without substrate and then assaying the free enzyme by the addition of substrate and following its hydrolysis for a period of time which is short relative to the dissociation rate of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal regulation of low density lipoprotein-sensitive events in a cholesterol transport mutant.

We have isolated and characterized Chinese hamster ovary cell mutants defective in the intracellular transport of low density lipoprotein (LDL)-derived cholesterol (Dahl, N. K., Reed, K. L., Daunais, M. A., Faust, J. R., and Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896). Mutant 2-2, which exhibits a cholesterol transport defect indistinguishable from the Niemann-Pick C phenotype, shows impaired but not absent LDL-mediated suppression of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. In parental cells, LDL suppression of HMG-CoA reductase is modulated by two mechanisms, decreased gene transcription and accelerated protein turnover. Using the chimeric protein HMGal as a reporter protein for LDL-mediated turnover and Northern blot analysis to monitor HMG-CoA reductase mRNA levels, we have dissected the contributions of these two regulatory responses to LDL-mediated suppression of HMG-CoA reductase activity. Kinetic modeling using the kinlsq program showed the following. Mutant 2-2 exhibits normal LDL-mediated acceleration of HMGal degradation, coupled with relatively abnormal regulation of mRNA. This suggests that the LDL-cholesterol signaling pathway to the nucleus is defective relative to the signal that results in HMG-CoA reductase turnover. In addition, LDL-mediated acceleration of HMGal turnover occurs well before LDL stimulation of cholesterol esterification in mutant 2-2, whereas these events occur synchronously in the parental cell line. This suggests that more than one pathway or mechanism exists for LDL-cholesterol signaling to the endoplasmic reticulum.

Kinlsq: a program for fitting kinetics data with numerically integrated rate equations and its application to the analysis of slow, tight-binding inhibition data.

Kinlsq, a Matlab-based computer program for the least-squares fitting of parameters to kinetics data described by numerically integrated rate equations, is described, and three applications to the analysis of enzyme kinetics data are given. The first application was to the analysis of a simple bimolecular enzyme plus inhibitor binding curve. The kinlsq fit to these data was essentially identical to that obtained with the corresponding analytically integrated rate equation, validating kinlsq. The second application was to the fit of a numerically integrated Michaelis-Menten model to the progress curve for dipeptidyl peptidase IV-catalyzed hydrolysis of Ala-Pro-p-nitroanilide as a demonstration of the analysis of steady-state enzyme kinetics data. The results obtained with kinlsq were compared with the results obtained by fitting this time course with the integrated Michaelis-Menten equation, and with the results obtained by fitting the (S,dP/dt) transform of the data with the Michaelis-Menten equation. The third application was to the analysis of the inhibition of chymotrypsin by the slow, tight-binding inhibitor MeOSuc-Ala-Ala-Pro-boroPhe, data not readily amenable to other methods of analysis. These applications demonstrate how kinlsq can be used to fit rate constants, equilibrium constants, steady-state constants, and the stoichiometric relationships between components.

Induction of glutathione-dependent formaldehyde dehydrogenase activity in Escherichia coli and Hemophilus influenza.

We have examined the induction of glutathione-dependent formaldehyde dehydrogenase (GS-FDH) activity in Escherichia coli and Hemophilus influenza. Formaldehyde was found to induce enzyme activity in both E. coli and H. influenza at concentrations between 0.6 and 20 ppm. Higher formaldehyde concentrations were toxic. Methanol concentrations up to 20% (200,000 ppm) and sodium formate concentrations up to 2% (20,000 ppm) gave negligible amounts of induction. The basic mechanism of induction was probed by inducing GS-FDH activity in the presence of rifampicin to inhibit RNA synthesis or chloramphenicol to inhibit protein synthesis. Both reagents inhibited GS-FDH induction, demonstrating that regulation occurs at the level of transcription. These results indicate that at least one function of GS-FDH in Gram-negative bacteria is to detoxify exogenous formaldehyde encountered in their environment and that GS-FDH inducibility may be a common feature of Gram-negative bacteria.

Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase.

The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized. It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates. Its isoelectric point, 4.4, is consistent with both its amino acid composition and chromatographic behavior on DEAE HPLC. The N-terminus is unblocked, and 47 residues from the N-terminus were sequenced. A computer search of the Swiss-Prot protein sequence data bank shows that the N-terminal sequence, [sequence; see text], is homologous with the mammalian class III alcohol dehydrogenases with 27 identities when compared to the human enzyme. Like the human, rat, and rabbit enzymes, it has high formaldehyde dehydrogenase activity in the presence of glutathione and catalyzes the oxidation of normal alcohols (ethanol, octanol, 12-hydroxydodecanoate) in a reaction that is not GSH-dependent. In addition, hemithiolacetals other than those formed from GSH, including omega-thiol fatty acids, also are substrates. The wide distribution and high degree of similarity of this enzyme to the plant and animal alcohol dehydrogenases suggest that the E. coli enzyme is closely related to the ancestor of the plant and animal dimeric zinc alcohol dehydrogenases.

Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays.

D-Alanine (D-Ala) is a ubiquitous constituent of bacterial cell walls. Assays for D-Ala can be used to investigate several aspects of cell wall biosynthesis and the effects of antibiotics on this process. High-sensitivity fluorescent assays for D-Ala were developed in a microtiter plate format based on d-aminoacid oxidase/horseradish peroxidase (DAO/HRP)-coupled reactions. For comparative purposes the classic chromogenic (UV-vis) assay using o-phenylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lower limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commercially available fluorogenic HRP substrates were then tested in this assay. Amplex Red (AR) gave a lower limit of sensitivity of 2 pmol and was linear up to 400 pmol d-Ala. QuantaBlu (QB) based assays exhibited a lag in their response to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calibration, but could be eliminated by addition of 150 pmol D-Ala to all assays. The QB assays were linear up to 3000 pmol D-Ala and gave a lower limit of sensitivity of 10 pmol. These assays are demonstrated for the characterization of the dd-carboxypeptidase activity of a soluble form of Escherichia coli penicillin-binding protein 5 (PBP 5) against the classic PBP substrate diacetyl-L-Lys-D-Ala-D-Ala. AR and QB based assays gave identical v/E(T) profiles, whereas OPD based assays gave slightly (10%) higher activity. This is consistent with the loss of a small amount of E. coli PBP 5 activity during the dilution necessary prior to its use in the highly sensitive fluorescent assays. These assays were then demonstrated for characterization of vancomycin binding to a D-Ala-D-Ala-based substrate.

15N and 1H NMR spectroscopy of the catalytic histidine in chloromethyl ketone-inhibited complexes of serine proteases.

The hemiketal hydroxyl groups in chloromethyl ketone (cmk) complexes of trypsin and chymotrypsin have been reported to ionize to the oxyanion with pK(a) values 2-4 pK(a) units below expectations for such a functional group on the basis of the behavior of the hemiketal carbon atom in 13C NMR spectra [Finucane, M. D., & Malthouse, J. P. G. (1992) Biochem. J. 286, 889-900]. The low pK(a) indicates the enzymes selectively stabilize the oxyanion form of the bound inhibitor, and therefore that cmk complexes may be good models of enzyme-mediated transition-state stabilization. However, the 13C NMR studies could not rule out His57 as the titrating group. Here we report the behavior of the ring 15N atoms of His57 in the Ala-Ala-Pro-Val-cmk complex of alpha-lytic protease. Both N(delta 1) and N(epsilon 2) of His57 respond to an ionization with a pK(a) of approximately 7.5, but His57 itself does not titrate as N(epsilon 2) remains alkylated and N(delta 1) remains bonded to a proton over the entire pH range. The species titrating with a pK(a) of approximately 7.5 must therefore be the hemiketal hydroxyl. The results also show that the 1H NMR signal from the proton in the Asp-His hydrogen bond behaves in a characteristic manner in cmk complexes and can be used diagnostically to confirm that His57 does not titrate and to measure the pK(a) of the hemiketal hydroxyl in cmk-protease complexes without resorting to 15N-labeling. We have used the behavior of this signal to directly confirm that His57 does not titrate in the trypsin and chymotrypsin complexes that were the subjects of the original 13C NMR studies.

Mechanism of HIV-1 Tat induced inhibition of antigen-specific T cell responsiveness.

HIV-1 Tat has been shown to have an inhibitory effect on the Ag-specific responsiveness of human peripheral T cells. We have previously demonstrated that this retroviral protein binds to and partially inhibits the enzymatic activity of dipeptidyl aminopeptidase type IV (DP IV), also known as CD26, which is expressed on a variety of mammalian tissue, including T lymphocytes. A number of studies have implicated a role for DP IV in the activation of T lymphocytes. By utilizing HIV-1 Tat, as well as ProboroPro, a potent and specific boronic acid analog inhibitor of DP IV, we show here that blocking DP IV partially inactivates Ag and anti-CD3-mediated T cell proliferation. Neither mitogen nor anti-CD2 mediated proliferation of T lymphocytes, however, is impaired by blocking DP IV. The target molecule for the inhibition induced by both compounds was confirmed by the finding that soluble DP IV neutralized the reduced Ag responsiveness. The Ag-specific inhibition could be overcome by the addition of exogenous IL-2, suggesting that blocking or inactivation of DP IV results in a state of anergy, probably by interfering with the delivery or amplification of a signal necessary for IL-2 production. This is further substantiated by the finding that costimulation of human PBMC via the CD28 molecule, which initiates a non-TCR-dependent signaling pathway, overcomes the reduced Ag responsiveness induced by Tat and ProboroPro. The fact that ProboroPro has no impact on stimulation of T cells with PMA and ionomycin implies that blocking DP IV is influencing events before the activation of protein kinase C and Ca2+ flux. These results suggest that DP IV is necessary for amplification of signals generated by the engagement of the TCR-CD3 complex by nominal Ag.

Solution structures of active and inactive forms of the DP IV (CD26) inhibitor Pro-boroPro determined by NMR spectroscopy.

Synthesis of the boronic acid analog of the dipeptide Pro-Pro yields a mixture of diastereomers Pro-L-boroPro and Pro-D-boroPro, one of which is a potent inhibitor [Ki = 16 pM; Gutheil, W. G., & Bachovchin, W. W. (1993) Biochemistry 32, 8723-8731] of dipeptidyl amino peptidase type IV (DP IV), also known as CD26. The structures of both diasteremers are determined here in aqueous solution by means of 1D and 2D NMR of 1H, 13C, and 11B, and force-field calculations, and the inhibitor is proven to have the L-L configuration. At low pH values (approximately 2), both diastereomers are trans with respect to the peptide bond. Populations of proline ring conformers are determined by pseudorotation analysis, using vicinal proton spin-coupling constants obtained by computer analysis of 1D1H NMR spectral fine structure. At neutral pH values, the Pro-boroPro inhibitor of DP IV undergoes slow, reversible inactivation (Gutheil & Bachovchin, 1993). By structural determination of the decomposition products of both diasteromers, the process is shown here to involve formation of a six-membered ring between the residues by means of trans-cis conversion and formation of a B-N bond, producing chiral nitrogen atoms in both cases having the S configuration. Analogy to cyclic dipeptides suggests the new compounds be named cyclo(Pro-L-boroPro) and cyclo(Pro-D-boroPro).

Human immunodeficiency virus 1 Tat binds to dipeptidyl aminopeptidase IV (CD26): a possible mechanism for Tat's immunosuppressive activity.

The human immunodeficiency virus 1 (HIV-1) Tat protein suppresses antigen-induced, but not mitogen-induced, activation of human T cells when added to T-cell cultures [Viscidi, R. P., Mayur, K., Lederman, H. M. & Frankel, A. D. (1989) Science 246, 1606-1608]. This activity is potentially pertinent to the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Here we report that Tat binds with high affinity to the T-cell activation molecule dipeptidyl aminopeptidase IV (DP IV), also known as CD26. This molecule occurs on the surface of CD4+ cells responsible for the recall antigen response and appears to play an essential role in this response. Tat binds to both the cell surface and soluble forms of DP IV at physiological salt concentrations without inhibiting the protease activity of DP IV against small chromogenic substrates used to assay activity, but Tat markedly inhibits the activity of DP IV at lower salt concentrations. The kinetics of inhibition indicate the affinity of Tat for DP IV varies from 20 pM to 11 nM, and the activity of the Tat-DP IV complex varies from 13% to 100%, as the NaCl concentration varies from 0 to 140 mM. Cytofluorometry experiments demonstrate that Tat competes with anti-Ta1, a monoclonal antibody (mAb) specific for DP IV, for binding to cell surface DP IV, thus indicating that Tat binds DP IV at or near the Ta1 epitope. Moreover, the anti-Ta1 mAb blocks the immunosuppressive activity of Tat. The high affinity of Tat for DP IV, previous evidence implicating DP IV in antigen-specific T-cell activation events, and the ability of anti-Ta1 mAb to block the immunosuppressive effect of Tat make DP IV a plausible receptor for Tat's immunosuppressive activity.