Study of aldose reductase inhibition in intact lenses by 13C nuclear magnetic resonance spectroscopy.

Carbon-13 nuclear magnetic resonance spectroscopy has been used in the study of glucose metabolism, specifically aldose reductase inhibition, in intact rabbit lenses maintained in organ culture. This technique provides an effective method of screening potential inhibitors of aldose reductase under conditions that more closely approximate in vivo conditions than do earlier methods. The aspirin substitutes acetaminophen and ibuprofen were studied as aldose reductase inhibitors and were found to be effective in reducing sorbitol accumulation in lenses exposed to high glucose stress. Results of this work with various inhibitors of aldose reductase are discussed in terms of lens metabolism and implications regarding diabetic complications such as cataract formation.

High expression and steady-state kinetic characterization of methionine site-directed mutants of Escherichia coli methionyl- and selenomethionyl-dihydrofolate reductase.

A high expression system that produces Escherichia coli dihydrofolate reductase (DHFR) at 30% total cellular protein was constructed. This expression vector, named pCOCK, allowed for the purification of nearly 100 mg of homogeneous DHFR from a 11 bacterial culture. A simple, single Q-Sepharose anion exchange column purification was developed on an FPLC instrument. Methionine site-directed mutants were constructed in DHFR to assess the role of Met within the enzymes. These mutants consisted of a Met16leucine (Leu), Met20Leu, Met42Leu, Met92Leu, Met16,20Leu and Met16,20,42Leu. Steady-state kinetic studies showed that the Met16Leu, Met42Leu and Met92Leu mutants possessed essentially the same kcat, Km(DHF) and Km(NADPH) as that of wild-type (wt) DHFR (13.7 s-1, 0.97 microM and 2.52 microM, respectively). Mutants which contained a Leu at position 20 possessed substantially elevated specific activity and kcat values. The specific activity and kcat of wt, Met20Leu, Met16,20Leu and Met16,20,42Leu were 45.9, 92.7, 90.2 and 172 mumol/min/mg and 13.7, 24.6, 25.2 and 52.7 s-1, respectively. Upon substitution of Met by selenomethionine (SeMet) in the aforementioned mutants, further information as to the effect of SeMet incorporation into proteins was ascertained. Steady-state kinetic parameters of the SeMet substituted Met16Leu, Met20Leu, Met42Leu and Met92Leu mutants were nearly identical to those of their Met containing counterparts. These data indicate that Met apparently has a limited role in the protein structure and function of DHFR and that SeMet incorporation has no effect on the steady-state kinetic constants of DHFR.

Muscarinic and nicotinic activities of the novel acetylcholine analog acetylselenonium choline.

The present paper further characterizes the cholinergic properties of acetylselenonium choline (ASeCh, (CH3)2Se+CH2CH2OCOCH3). The data demonstrate that ASeCh possesses muscarinic receptor agonist properties as evidenced by vasodepressor and smooth muscle contractile activities which are enhanced by physostigmine and antagonized by atropine. ASeCh also possessed nicotinic agonist activity on frog rectus abdominis tissue which was potentiated by physostigmine, and blocked by d-tubocurarine. The relative potencies of ASeCh ranged from approximately 1% to approximately 6% of the potency of acetylcholine in the three types of preparations examined.

Synthesis of novel tellurium containing analogues of choline and acetylcholine and their quantitation by pyrolysis-gas chromatography-mass spectrometry.

Methods for the synthesis and quantitation of the novel choline analogues, telluronium choline and acetyltelluronium choline, are described. An assay procedure utilizing pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) with cold trapping was developed with [2H4]telluronium choline and [2H4]acetyltelluronium choline as internal standards. The telluronium compounds were ion-pair extracted from tissue with dipicrylamine, washed with 2-butanone, and pyrolyzed prior to GC-MS analysis. The compounds were monitored using selected ion monitoring at m/z 232 and m/z 190 for acetyltelluronium and telluronium choline, respectively, and at m/z 236 and m/z 194 for the analogous deuterated internal standards. The assay was linear over a range of 20 pmol-20 nmol of compound taken through the assay.

The utilization of 13C and 31P nuclear magnetic resonance spectroscopy in the study of the sorbitol pathway and aldose reductase inhibition in intact rabbit lenses.

Nuclear magnetic resonance (NMR) spectroscopy has been utilized in the study of the metabolism of intact, functioning rabbit lenses maintained in organ culture. The sorbitol pathway and aldose reductase inhibition have been studied using carbon-13 NMR spectroscopy. Incubation of lenses in high concentration [1-13C] glucose medium with and without added inhibitors allows the sorbitol pathway and glycolysis to be monitored. Various aldose reductase inhibitors have been studied and are ranked based on percentage of inhibition as follows: tolrestat greater than or equal to sorbinil greater than sulindac greater than sulindac sulfide much greater than indomethacin greater than acetylsalicylic acid greater than quercetin greater than tandearil greater than salicylic acid greater than 3,3-Tetramethyleneglutaric acid (TMG). It has been demonstrated that 13C NMR spectroscopy provides an effective method of screening potential inhibitors of aldose reductase. The aspirin substitutes ibuprofen and acetaminophen have been studied and are found to reduce sorbitol accumulation in intact rabbit lenses. The effects of myo-inositol and vitamin E on sorbitol accumulation have also been investigated. Results suggest that the various metabolic pathways within the lens are intricately connected. In a preliminary manner, the effect of diabetes on metabolism in intact lenses has been investigated using 13C NMR spectroscopy. Increased sorbitol production has been observed for diabetic lenses. 31P NMR spectroscopy has also been utilized in the study of lens metabolism and aldose reductase inhibitors. Inclusion of various inhibitors in the high concentration glucose medium results in maintenance of essentially normal phosphorus-containing metabolite levels in the lens. No clear relationship was observed between lens clarity and phosphorus metabolite levels as determined using NMR.

Synthesis and characterization of a selenium-containing substrate of alpha-chymotrypsin. Selenium-77 nuclear magnetic resonance observation of an acyl-alpha-chymotrypsin intermediate.

The selenium-containing ester p-nitrophenyl (phenylselenyl)acetate, C6H5SeCH2C(O)-OC6H4-p-(NO2), has been synthesized, characterized as a substrate for alpha-chymotrypsin (k2/KM = 15.2 X 10(3) M-1 s-1, KMapp = 5.16 X 10(-6) M, pH 7.77, 33% CH3CN, 25 degrees C), and shown to be an active-site titrant for the enzyme. A synthesis of the selenium-77 enriched p-nitrophenyl (phenylselenyl)acetate in 53% yield from 94.4% elemental selenium-77, followed by its reaction with alpha-chymotrypsin (pH 5.0, 0-3 degrees C), permitted the observation of the (phenylselenyl)acetyl-alpha-chymotrypsin reaction intermediate by selenium-77 NMR spectroscopy. This acyl-enzyme species had a chemical shift of 275.1 ppm relative to dimethyl selenide. Accompanying this resonance was a lower intensity, pH-dependent resonance that is assigned to (phenylselenyl)acetate on the basis of a pH titration of the model compound. Deacylation in the presence of hydrazine sulfate produced a resonance at 332.3 ppm in addition to the 302.2 ppm resonance of (phenylselenyl)acetate at pH 7.85. Denaturation of the acyl-enzyme resulted in a shift of the 275.1 ppm resonance to 334.6 ppm at pH 4.90, in good agreement with the selenium-77 chemical shift of the model compound, methyl (phenylselenyl)acetate, in CDCl3 (333.3 ppm). The large shielding observed for the native acyl-enzyme in comparison to the denatured species can be attributed to a resonance-perturbed ester linkage and/or steric compression at a nonbonding orbital of the selenium nucleus.

Increased lactate dehydrogenase isoenzyme 1 in serum and tumor tissue of a patient with small-cell carcinoma.

Histological examination of supraclavicular lymph node tissue obtained at biopsy from a 63-year-old man disclosed metastatic small-cell carcinoma. On admission and for four days subsequently, total lactate dehydrogenase (LD; EC 1.1.1.27) activity in serum was 6.5 times normal; studies of LD isoenzyme showed persistently increased LD-1, with LD-1 greater than LD-2. Isoenzyme electrophoresis of tissue homogenates prepared from the patient's tumor also showed the LD-1 greater than LD-2 pattern. Isoenzyme studies for supraclavicular lymph node tissue from five control subjects showed contrasting isoenzyme patterns as compared with the patients in whom LD-2, LD-3, and LD-4 predominated. Because these abnormalities were persistent, they differ from the temporal sequence for LD usually seen in myocardial infarction. This emphasizes the importance of repetitive sampling for clinical interpretation of data on this enzyme.

1H NMR spectroscopic studies of selenosubtilisin.

Anomalously low-field signals in 1H NMR spectra of serine proteases provide valuable information on the protonation state of the catalytic histidine residue. We have examined the pH dependence of the deshielded protons of three different oxidation states of selenosubtilisin, a semisynthetic selenoenzyme with significant peroxidase activity, in order to evaluate the influence of the selenium prosthetic group on the hydrogen-bonding network in the modified active site. In the spectra of the anionic seleninate and selenolate derivatives, two resonances were observed at 18.0 and 15.5/14.0 ppm, assigned respectively to the N delta 1 and N epsilon 2 protons of protonated His64. These signals were apparent from pH 4 to above pH 10, indicating that the negatively charged prosthetic group increases the stability of the imidazolium dramatically, raising its pKa by at least 3-4 pH units. In contrast, a neutral selenenyl sulfide species exhibits no deshielded proton signals at 18 ppm at any pH but has a weak signal at 14.1 ppm above pH 7 which was assigned to the N delta 1 imidazole proton of neutral His64. While the pKa of His64 appears normal (approximately 7) in this derivative, the selenenyl sulfide substitution may alter the orientation of the imidazole ring within the active site for steric reasons. Together with data on the influence of pH on peroxidase activity, these results suggest that selenosubtilisin's His64 acts as a general acid facilitating the reduction of the selenenyl sulfide to selenolate by thiols.

Telluromethionine in structural biochemistry.

One of the fundamental problems in macromolecular crystallography is the availability of the suitable heavy-atom derivatives necessary to solve the phase problem. The ability to label a protein with a tellurium-containing amino acid (telluromethionine) at internal sites through the utilization of protein biosynthesis supplies x-ray crystallographers a convenient phasing vehicle and nuclear magnetic resonance (NMR) spectroscopists an internal probe with which to study structure/function relationships via Te-125 NMR spectroscopy. In this communication we demonstrate the partial incorporation of telluromethionine into E. coli dihydrofolate reductase (DHFR) with no apparent perturbations to activity or substrate binding. Enzyme containing two moles TeMet exhibited a specific activity of 42 units/mg and a 1:1 binding ratio with methotrexate.

Demonstration of the feasibility of observing nuclear magnetic resonance signals of 77Se covalently attached to proteins.

Previous 77Se NMR relaxation time studies established the utility of 77Se NMR spectroscopy in studying low molecular weight (less than 500) selenium-containing molecules. Since the spin rotation and chemical shift anisotrophy mechanisms contributed significantly to the 77Se spin-lattice relaxation in these compounds, it was questionable as to whether the latter mechanism would be efficient enough to enable 77Se resonances to be observed in a reasonable period in high molecular weight selenobiomolecules. Thus, to address this problem, disulfide bonds of ribonuclease-A and lysozyme were reductively cleaved under denaturing conditions, and the resulting 7-8 sulfhydryl groups were treated with a new sulfhydryl group reagent containing selenium, 6,6'-diselenobis(3-nitrobenzoic acid), to give proteins containing covalently attached selenium in the form of selenenyl sulfides. The observation of high resolution 77Se NMR spectra of these proteins under denaturing conditions was accomplished. Five to six 77Se NMR resonances, which fell in a chemical shift range of 14-15 ppm, were observed for each protein and are compared to the chemical shifts of several model selenenyl sulfides derived from cysteine.

Expression, characterization and crystallographic analysis of telluromethionyl dihydrofolate reductase.

Selenomethionine-containing proteins analyzed by multi-wavelength anomalous diffraction provide a facile means of addressing the phase problem, whose solution is necessary to determine protein structures by X-ray crystallography [Hendrickson (1991). Science, 254, 51-58]. Since this method requires synchrotron radiation, we sought to incorporate a true heavy atom into protein, allowing the solution of the phase problem by more traditional methods of data collection. Media containing TeMet alone or TeMet with low levels of Met failed to sustain growth of a methione auxotroph of Escherichia coli carrying the dihydrofolate reductase expression vector. Growth of the organism to stationary phase and incorporation of TeMet was observed when the culture was initiated in media containing minimal Met levels and TeMet was added after induction with isopropyl-1-thio-beta-D-galactopyranoside. The purified enzyme exhibited properties similar to those of the native enzyme. Atomic absorption spectroscopy and amino-acid analysis indicated that 40% of the methionines were replaced with TeMet. Sequence analysis did not indicate significant levels of replacement in the first three sites (1, 16 and 20), suggesting that TeMet was present only in the last two sites (42 and 92). Crystals of this enzyme were grown in the presence of methotrexate and were isomorphous with crystals of wild-type dihydrofolate reductase. Difference Fourier maps and restrained least-squares refinement showed no substitution at the first three methionines, while incorporation was seen at positions 42 and 92.

(R,R)-N,N'-dimethylcyclohexyl-1,2-diazaseleno-phospholidine as a chiral derivatizing agent for the evaluation of chiral alcohols.

Previously, a diazaphospholidine has been synthesized and evaluated as a chiral derivatizing reagent for the determination of the optical purity of chiral alcohols via 31P NMR spectroscopy (Alexakis et al., J. Org. Chem. 57:1224-1237, 1992). Our laboratory is interested in the advantageous and practical applications of 77Se NMR spectroscopic studies in many facets of chemistry and biochemistry. To this end we have used this diazaphospholidine as a starting point and have investigated chiral alcohols coupled to an optically pure diazaselenophospholidine. The diastereomers formed were then evaluated by 77Se NMR spectroscopy, and these results were compared to the 31P NMR results published by Alexakis and co-workers. It was found that addition of the Se atom produced diastereomers that were air stable and, in many cases, the individual diastereomers could be distinguished by 77Se NMR spectroscopy. Preliminary results indicate that the 77Se nucleus is somewhat more sensitive to remotely disposed chiral centers than is the 31P nucleus. Furthermore, because of their stability, these compounds do not readily decompose and can, therefore, be studied by a variety of chromatographic and spectroscopic techniques.

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme.

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.

Further biological activities of the novel choline analogs selenonium choline and acetylselenonium choline.

The pharmacological actions of the novel choline analog, selenonium choline [(CH3)2Se+CH2CH2OH] and its acetyl ester acetylselenonium choline (ASeCh) were studied in vivo and in vitro. ASeCh produced a dose-related decrease in mean arterial pressure in the rat similar to acetylcholine (ACh) but was 1% to 2% as potent. ASeCh demonstrated agonist activity on the rat isolated ileum and was approximately 2% as active as ACh. Selenonium chlorine (SeCh) was taken up and acetylated in brain tissue slices in a time- and concentration-dependent manner. The use of KCl as a loading stimulus did not increase the uptake of SeCh but increased tissue levels of ASeCh 1.5-fold over the control concentrations. The uptake of SeCh was described by a single low-affinity uptake component (Km = 167 microM) that was not blocked by hemicholinium-3. In contrast, hemicholinium significantly blocked the acetylation of SeCh. Compared with basal release, depolarization with KCl caused a significant release of ASeCh into the incubation medium. A neural specificity was suggested for the in vitro uptake of SeCh. Acetylation of SeCh in vivo in the rat after intraventricular administration was similar to the extent of acetylation of [2H4]-choline. ASeCh bound to both M1 and M2 cholinergic receptors with 2% to 3% of the affinity observed for ACh. These data suggest that SeCh may satisfy criteria for a false neurotransmitter precursor.

Selenomethionyl dihydrofolate reductase from Escherichia coli. Comparative biochemistry and 77Se nuclear magnetic resonance spectroscopy.

The biosynthetic replacement of Met residues by selenomethionine (SeMet) facilitates the determination of three-dimensional structure by multiwavelength anomalous diffraction (Yang, W., Hendrickson, W. A., Crouch, R.J., and Satow, Y. (1990) Science 249, 1398-1405). In an effort to examine any biochemical effects due to the replacement of Met residues by SeMet, we chose to compare the kinetic and binding properties of selenomethionyl dihydrofolate reductase with those of the wt enzyme. There are 5 Met residues in Escherichia coli dihydrofolate reductase with 2 located in the Met-20 loop, which is a sequence of residues forming a lid over the active site. Utilizing plasmid pWT8, which affords 10-15% soluble protein as E. coli dihydrofolate reductase, we readily isolated both the SeMet and wt enzymes from E. coli DL41 utilizing a novel purification protocol. Both enzymes exhibited essentially the same kinetic and binding properties, including specific activities (45 mumol/min/mg), Km (7,8-dihydrofolate = 0.39 microM; NADPH = 2.0 microM), kcat (13.5/s), and 1:1 noncovalent inhibitory binding ratios with methotrexate. The inhibitory effects of divalent and monovalent cations on activity were also assessed, with the SeMet-containing enzyme exhibiting a uniformly greater sensitivity than the wt enzyme. We conclude that the biochemical properties of dihydrofolate reductase are virtually unperturbed by SeMet inclusion. Analysis of SeMet dihydrofolate reductase by 77Se nuclear magnetic resonance spectroscopy revealed five distinct resonances, thus indicating the potential value of this technique in employing selenium as a nonperturbing NMR probe of protein structure and function.

Selected biological activities of novel selenonium choline analogs.

1. The novel choline analogs selenonium choline (SeCh) and acetylselenonium choline (ASeCh) have been examined for selected biological activities. 2. ASeCh was found to be an alternative substrate for acetylcholine esterase with Km and Vmax values similar to acetylcholine. 3. ASeCh and SeCh inhibited acetylthiocholine hydrolysis by acetylcholinesterase with IC50 values similar to acetylcholine and choline. 4. SeCh exerted a protective action against physostigmine and DFP induced toxicity. 5. SeCh (85 mg/kg) was found to be 3 times more toxic in mice than choline.

The crystal structure of N(10)-formyltetrahydrofolate synthetase from Moorella thermoacetica.

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.

Cation binding and thermostability of FTHFS monovalent cation binding sites and thermostability of N10-formyltetrahydrofolate synthetase from Moorella thermoacetica.

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.