Alkaptonuria.
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4-Hydroxyphenylpyruvate Dioxygenase↗
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Natural history of alkaptonuria.
BACKGROUND: Alkaptonuria, caused by mutations in the HGO gene and a deficiency of homogentisate 1,2-dioxygenase, results in an accumulation of homogentisic acid (HGA), ochronosis, and destruction of connective tissue. There is no effective therapy for this disorder, although nitisinone inhibits the enzyme that produces HGA. We performed a study to delineate the natural history of alkaptonuria. METHODS: We evaluated 58 patients with alkaptonuria (age range, 4 to 80 years), using clinical, radiographic, biochemical, and molecular methods. A radiographic scoring system was devised to assess the severity of spinal and joint damage. Two patients were treated with nitisinone for 10 and 9 days, respectively. RESULTS: Life-table analyses showed that joint replacement was performed at a mean age of 55 years and that renal stones developed at 64 years, cardiac-valve involvement at 54 years, and coronary-artery calcification at 59 years. Linear regression analysis indicated that the radiographic score for the severity of disease began increasing after the age of 30 years, with a more rapid increase in men than in women. Twenty-three new HGO mutations were identified. In a 51-year-old woman, urinary HGA excretion fell from 2.9 to 0.13 g per day after a 10-day course of nitisinone (7 days at a dose of 0.7 mg per day and 3 days at 2.8 mg per day). In a 59-year-old woman, urinary HGA fell from 6.4 g to 1.7 g per day after nine days of treatment with nitisinone (0.7 mg per day). Plasma tyrosine levels in these patients rose from approximately 1.1 mg per deciliter (60 micromol per liter) in both to approximately 12.8 mg per deciliter (700 micromol per liter) and 23.6 mg per deciliter (1300 micromol per liter), respectively, with no clinical signs or symptoms. CONCLUSIONS: The reported data on the natural history of alkaptonuria provide a basis for the evaluation of long-term therapies. Although nitisinone can reduce HGA production in humans with homogentisate 1,2-dioxygenase deficiency, the long-term safety and efficacy of this treatment require further evaluation.
4-Hydroxyphenylpyruvate Dioxygenase↗
Propagation failures, breathing pulses, and backfiring in an excitable reaction-diffusion system.
We report results from experiments with a pseudo-one-dimensional Belousov-Zhabotinsky reaction that employs 1,4-cyclohexanedione as its organic substrate. This excitable system shows traveling oxidation pulses and pulse trains that can undergo complex sequences of propagation failures. Moreover, we present examples for (i) breathing pulses that undergo periodic changes in speed and size and (ii) backfiring pulses that near their back repeatedly generate new pulses propagating in opposite direction.
Animals↗
New biphenol-based, fine-tunable monodentate phosphoramidite ligands for catalytic asymmetric transformations.
Monodentate phosphoramidite ligands have been developed based on enantiopure 6,6'-dimethylbiphenols with axial chirality. These chiral ligands are easy to prepare and flexible for modifications. The fine-tuning capability of these ligands plays a significant role in achieving high enantioselectivity in the asymmetric hydroformylation of allyl cyanide and the conjugate addition of diethylzinc to cycloalkenones.
Allyl Compounds↗
Widespread sulfenic acid formation in tissues in response to hydrogen peroxide.
A principal product of the reaction between a protein cysteinyl thiol and hydrogen peroxide is a protein sulfenic acid. Because protein sulfenic acid formation is reversible, it provides a mechanism whereby changes in cellular hydrogen peroxide concentration may directly control protein function. We have developed methods for the detection and purification of proteins oxidized in this way. The methodology is based on the arsenite-specific reduction of protein sulfenic acid under denaturing conditions and their subsequent labeling with biotin-maleimide. Arsenite-dependent signal generation was fully blocked by pretreatment with dimedone, consistent with its reactivity with sulfenic acids to form a covalent adduct that is nonreducible by thiols. The biotin tag facilitates the detection of protein sulfenic acids on Western blots probed with streptavidin-horseradish peroxidase and also their purification by streptavidin-agarose. We have characterized protein sulfenic acid formation in isolated hearts subjected to hydrogen peroxide treatment. We have also purified and identified a number of the proteins that are oxidized in this way by using a proteomic approach. Using Western immunoblotting we demonstrated that a highly significant proportion of some individual proteins (68% of total in one case) form the sulfenic derivative. We conclude that protein sulfenic acids are widespread physiologically relevant posttranslational oxidative modifications that can be detected at basal levels in healthy tissue, and are elevated in response to hydrogen peroxide. These approaches may find widespread utility in the study of oxidative stress, particularly because hydrogen peroxide is used extensively in models of disease or redox signaling.
Animals↗
Potency of Michael reaction acceptors as inducers of enzymes that protect against carcinogenesis depends on their reactivity with sulfhydryl groups.
Induction of phase 2 enzymes and elevations of glutathione are major and sufficient strategies for protecting mammals and their cells against the toxic and carcinogenic effects of electrophiles and reactive forms of oxygen. Inducers belong to nine chemical classes and have few common properties except for their ability to modify sulfhydryl groups by oxidation, reduction, or alkylation. Much evidence suggests that the cellular "sensor" molecule that recognizes the inducers and signals the enhanced transcription of phase 2 genes does so by virtue of unique and highly reactive sulfhydryl functions that recognize and covalently react with the inducers. Benzylidene-alkanones and -cycloalkanones are Michael reaction acceptors whose inducer potency is profoundly increased by the presence of ortho- (but not other) hydroxyl substituent(s) on the aromatic ring(s). This enhancement correlates with more rapid reactivity of the ortho-hydroxylated derivatives with model sulfhydryl compounds. Proton NMR spectroscopy provides no evidence for increased electrophilicity of the beta-vinyl carbons (the presumed site of nucleophilic attack) on the hydroxylated inducers. Surprisingly, these ortho-hydroxyl groups display a propensity for extensive intermolecular hydrogen bond formation, which may raise the reactivity and facilitate addition of mercaptans, thereby raising inducer potencies.
Animals↗
An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors.
cDNA fragments encoding the carboxyltransferase domain of the multidomain plastid acetyl-CoA carboxylase (ACCase) from herbicide-resistant maize and from herbicide-sensitive and herbicide-resistant Lolium rigidum were cloned and sequenced. A Leu residue was found in ACCases from herbicide-resistant plants at a position occupied by Ile in all ACCases from sensitive grasses studied so far. Leu is present at the equivalent position in herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases containing a 1000-aa fragment of two ACCase isozymes found in a herbicide-resistant maize were expressed in a yeast ACC1 null mutant to test herbicide sensitivity of the enzyme in vivo and in vitro. One of the enzymes was resistant/tolerant, and one was sensitive to haloxyfop and sethoxydim, rendering the gene-replacement yeast strains resistant and sensitive to these compounds, respectively. The sensitive enzyme has an Ile residue, and the resistant one has a Leu residue at the putative herbicide-binding site. Additionally, a single Ile to Leu replacement at an equivalent position changes the wheat plastid ACCase from sensitive to resistant. The effect of the opposite substitution, Leu to Ile, makes Toxoplasma gondii apicoplast ACCase resistant to haloxyfop and clodinafop. In this case, inhibition of the carboxyltransferase activity of ACCase (second half-reaction) of a large fragment of the Toxoplasma enzyme expressed in Escherichia coli was tested. The critical amino acid residue is located close to a highly conserved motif of the carboxyltransferase domain, which is probably a part of the enzyme active site, providing the basis for the activity of fop and dim herbicides.
Acetyl-CoA Carboxylase↗
Two-dimensional IR spectroscopy can be designed to eliminate the diagonal peaks and expose only the crosspeaks needed for structure determination.
The power of two-dimensional (2D) IR spectroscopy as a structural method with unprecedented time resolution is greatly improved by the introduction of IR polarization conditions that completely eliminate diagonal peaks from the spectra and leave only the crosspeaks needed for structure determination. This approach represents a key step forward in the applications of 2D IR to proteins, peptides, and other complex molecules where crosspeaks are often obscured by diagonal peaks. The technique is verified on the model compound 1,3-cyclohexanedione and subsequently used to clarify the distribution of structures that the acetylproline-NH(2) dipeptide adopts in chloroform. In both cases, crosspeaks are revealed that were not observed before, which, in the case of the dipeptide, has led to additional information about the structure of the amino group end of the peptide.
Chloroform↗
Selective killing of transformed baby hamster kidney (BHK) cells.
We report here that certain drugs can protect Syrian baby hamster kidney cells (BHK) in culture against the lethal agents cytosine arabinonucleoside, hydroxyurea, and colcemid. Polyoma virus-transformed BHK cells (PyBHK) are killed under the same conditions. The protective drugs include caffeine and streptovitacin A. Kinetic studies show that these drugs act specifically in G1, and that they shift BHK cells from G1 into the G0 state at the restriction point, similar to the effects of high cell density or serum deprivation. These drugs do not block the growth of PyBHK cells nearly as effectively, consistent with a reduced effectiveness of restriction point control in virus-transformed cells. Consequently, the transformed cells around their cycle and are killed by the cell cycle phase-specific toxic agents, in contrast to the arrested BHK cells. These findings provide a model for studies on differential killing of tumor versus normal cells in vivo.
Antibiotics, Antineoplastic↗
Inhibition of receptor-mediated clearance of lysine and arginine-modified lipoproteins from the plasma of rats and monkeys.
Reductive methylation of at least 30% of the lysine residues or 1,2-cyclohexanedione modification of 45% of the arginine residues prevented low density lipoproteins (LDL) from binding to cell surface receptors of fibroblasts in vitro, without significantly altering other physical or chemical properties of the LDL. When rat or human LDL with more than 30% of the lysine residues methylated were injected intravenously into rats, the clearance of these lipoproteins from the plasma was slowed considerably. The half-life of the reductively methylated LDL was approximately twice that obtained for control (unmodified) LDL, and the value for the fractional catabolic rate was approximately half that of the control. Furthermore, when human LDL modified by reductive methylation were injected into rhesus monkeys, the rate of clearance was similarly retarded, and the value for the fractional catabolic rate was reduced by approximately 50% as compared with the value for control LDL. A dual isotope labeling technique ((125)I and (131)I) was used to compare the disappearance of the control and modified LDL in the same animal. It was demonstrated that not only modification of lysine residues but also modification of the arginine residues with 1,2-cyclohexanedione retarded the plasma clearance of the rat LDL. However, the cyclohexanedione modification was spontaneously reversible at 37 degrees C, whereas reductive methylation of the lysine residues was stable. It is concluded that the selective chemical modification of lysine or arginine residues of LDL interferes with the normal uptake of these lipoproteins in vivo as well as by fibroblasts in vitro. These data provide an estimation of the level of receptor-mediated clearance of LDL from the plasma, a value that may be as high as 50% in rats and monkeys.
Animals↗
Relationship of biosynthesis of slow reacting substance to intracellular glutathione concentrations.
To further elucidate the role of glutathione (GSH) in the biosynthesis of slow reacting substance (SRS), SRS generation was studied in rat basophilic leukemia cells that had been preincubated with 2-cyclohexen-1-one or diethyl maleate to decrease their intracellular GSH concentrations. At low GSH levels SRS formation was markedly inhibited. The formation of other lipoxygenase products was much less affected, although some decrease in 5-hydroxyicosatetraenoic acid formation also occurred, apparently due in part to less rapid reduction of the 5-hydroperoxide.
Animals↗
Angiogenin stimulates endothelial cell prostacyclin secretion by activation of phospholipase A2.
Angiogenin stimulates capillary and umbilical vein endothelial cell prostacyclin secretion but not that of prostaglandins of the E series. The response was quantitated by radioimmunoassay and by [3H]arachidonate labeling followed by analysis of the secreted prostaglandins. The stimulated secretion lasts for several minutes and is optimal at 2-4 min. The dose-response (peak at 1-10 ng/ml) is similar to that previously observed for activation of endothelial cell phospholipase C. Stimulated secretion was blocked by pretreatment with the inhibitors of prostacyclin synthesis, indomethacin and tranylcypromine, and also the specific inhibitor of phospholipase A2, quinacrine, as well as pertussis toxin and the diglyceryl and monoglyceryl lipase inhibitor RHC 80267. Stimulated secretion was also abolished in cells that were either pretreated for 48 hr with phorbol ester to down-regulate protein kinase C or incubated with the protein kinase inhibitor H7. Hydrolysis of phosphatidylinositol by phospholipase A2 appears to be the source of angiogenin-mobilized arachidonate; angiogenin-induced hydrolysis of phosphatidylcholine was not detected. Activation of phospholipase A2 occurs in the absence of an angiogenin-induced calcium flux. The results are discussed in terms of mechanisms of agonist-induced intracellular arachidonate mobilization and relevance to angiogenesis.
Angiogenesis Inducing Agents↗
Dominant mutations causing alterations in acetyl-coenzyme A carboxylase confer tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides in maize.
A partially dominant mutation exhibiting increased tolerance to cyclohexanedione and aryloxyphenoxypropionate herbicides was isolated by exposing susceptible maize (Zea mays) tissue cultures to increasingly inhibitory concentrations of sethoxydim (a cyclohexanedione). The selected tissue culture (S2) was greater than 40-fold more tolerant to sethoxydim and 20-fold more tolerant to haloxyfop (an aryloxyphenoxypropionate) than the nonselected wild-type tissue culture. Regenerated S2 plants were heterozygous for the mutant allele and exhibited a high-level, but not complete, tolerance to both herbicides. Homozygous mutant families derived by self-pollinating the regenerated S2 plants exhibited no injury after treatment with 0.8 kg of sethoxydim per ha, which was greater than 16-fold the rate lethal to wild-type plants. Acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) is the target enzyme of cyclohexanedione and aryloxyphenoxypropionate herbicides. ACCase activities of the nonselected wild-type and homozygous mutant seedlings were similar in the absence of herbicide. ACCase activity from homozygous tolerant plants required greater than 100-fold more sethoxydim and 16-fold more haloxyfop for 50% inhibition than ACCase from wild-type plants. These results indicate that tolerance to sethoxydim and haloxyfop is controlled by a partially dominant nuclear mutation encoding a herbicide-insensitive alteration in maize ACCase.
Acetyl-CoA Carboxylase↗
A structural assignment for a stable acetaldehyde-lysine adduct.
Acetaldehyde is the first oxidation product of ethanol in vivo. Lysine residues in proteins such as hemoglobin have been implicated as target structures for acetaldehyde adducts resulting from ethanol consumption. Although the presence of both stable and unstable acetaldehyde-hemoglobin adducts has been established, the structural characterization of the adducts has received relatively little attention. As a model for such adduct formation, we studied the peptide pentalysine in vitro. Pentalysine has several potential sites for adduct formation. The amino-terminal amine group as well as the epsilon-amine groups of each lysine side chain can serve as potential sites for modification by acetaldehyde. Mass spectrometry, nuclear magnetic resonance, and Raman spectroscopy were employed to demonstrate that acetaldehyde forms a stable linkage to lysine amine groups via a Schiff base.
Acetaldehyde↗
A new old yellow enzyme of Saccharomyces cerevisiae.
In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V. (1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.
Amino Acid Sequence↗
Quantitating direct chlorine transfer from enzyme to substrate in chloroperoxidase-catalyzed reactions.
Substrate competition methods that were previously used to quantitate the involvement of free Cl2 in the chloride-dependent peroxidatic reactions catalyzed by chloroperoxidase (CPO) (Libby, R. D., Shedd, A. L., Phipps, K. A., Beachy, T. M., and Gerstberger, S. M., (1992) J. Biol. Chem. 267, 1769-1775) are extended to CPO-catalyzed halogenation reactions. Relative substrate specificities of halogen acceptor substrates (RHs) antipyrine (ap), NADH, 2-chlorodimedone (2cd), and barbituric acid (ba) are compared with previously studied peroxidatic substrates catechol (cat) and 2, 4,6-trimethylphenol (tmp) in their reactions with the CPO-H2O2-Cl system versus the hypochlorite-Cl system. Studies were carried out at pH 2.75 over a chloride concentration range of 1-100 mM and at pH 4.80 over a chloride concentration range of 100-400 mM. Competition studies involved successive pairwise comparisons of substrates of increasing enzyme specificity. The orders of specificities, ba > 2cd > ap > cat > tmp at pH 2.75 and ba > 2cd > NADH > ap > cat > tmp at pH 4.80, are the same for both the CPO-H2O2-Cl and hypochlorite-Cl systems. However, the magnitudes of the specificities are different between the two systems. In all comparisons except ap versus cat, the specificity of the CPO-H2O2-Cl system toward the preferred substrate is higher than that of the hypochlorite-Cl system. Quantitative comparisons between specificities of CPO-H2O2-Cl and hypochlorite-Cl systems indicate that at least 98% of the CPO-catalyzed halogenation reactions of ba, 2cd, NADH, and ap occur by mechanisms in which the substrate reacts directly with the enzyme. Thus, less than 2% of any of the CPO reactions could possibly involve a free oxidized halogen intermediate. All data are consistent with a mechanism in which RH binds to the CPO chlorinating intermediate (EOCl), and the chlorine atom is transferred directly from EOCl to RH. Further, the results indicate that any halogenation substrate with a higher CPO specificity than ap must also undergo direct chlorine transfer from the enzyme. These results underscore the critical need for quantitative kinetic evidence in establishing the extent of involvement of any potential reaction intermediate. Finally, this work calls into question the long held assumption of the obligatory involvement of hypochlorite as an intermediate in myeloperoxidase reactions. It supports the recent kinetic evidence presented by Marquet and Dunford for direct chlorine transfer in myeloperoxidase-catalyzed chlorination of tuarine (Marquet, L. A., and Dunford, H. B. (1994) J. Biol. Chem. 269, 7950-7956).
Antipyrine↗
Identification of arginyl residues located at the ATP binding site of sarcoplasmic reticulum Ca2+-ATPase. Modification with 1,2-cyclohexanedione.
Sarcoplasmic reticulum vesicles were treated with 1, 2-cyclohexanedione (CHD) in sodium borate (pH 8.0). The Ca2+-ATPase activity was completely inhibited. Inhibition of Mg.ATP and Mg.ADP binding to the high affinity ATP binding site as well as inhibition of phosphorylation with ATP occurred simultaneously with the inhibition of the Ca2+-ATPase activity. Phosphorylation with acetyl phosphate was not inhibited. The Ca2+-ATPase was strongly protected by Mg.ATP, Mg.ADP, and Mg.AMP against this inhibition. Binding of acetyl phosphate or Pi to the enzyme gave no protection. Phosphorylation with acetyl phosphate also had no protective effect. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, and sequencing revealed that Arg-489, Arg-505, and Arg-678 were modified with CHD. Arg-489 and Arg-678 were almost completely protected by Mg.ATP against this modification, but partially protected by prelabeling with fluorescein 5-isothiocyanate, which occupies the adenosine binding region in the ATP binding site. In contrast, Arg-505 was slightly protected by Mg.ATP and almost completely protected by prelabeling with fluorescein 5-isothiocyanate. Taken together, these findings suggest that Arg-489 and Arg-678 are located in or near the region occupied by the triphosphate moiety of ATP, either or both of these residues being in or close to the region occupied by the alpha-phosphoryl group in the high affinity ATP binding site and involved in the CHD-induced inhibition of this enzyme and that Arg-505 is very close to (but slightly out of) the adenosine binding region in the ATP binding site. The acetyl phosphatase activity and phosphorylation with Pi were also inhibited by the CHD treatment, but the inhibitions were considerably slower than those described above. This suggests that the arginyl residues involved in these inhibitions are distinct from that involved in the inhibition of the Ca2+-ATPase activity.
Adenosine Triphosphate↗
Modification of arginine-198 in sarcoplasmic reticulum Ca2+-ATPase by 1,2-cyclohexanedione causes inhibition of formation of the phosphoenzyme intermediate from inorganic phosphate.
Sarcoplasmic reticulum vesicles were modified with 1,2-cyclohexanedione (CHD), a specific arginine-modifying reagent, in sodium borate (pH 8.0 or 8.8). Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by the modification with CHD. Tight binding of F- and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, either of which produces a transition state analog for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also markedly inhibited. In contrast, phosphoenzyme formation from acetyl phosphate in the forward reaction was unaffected. The enzyme was appreciably protected by tight binding of F- and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+, but not by the presence of 20 mM MgCl2 alone or 150 mM Pi alone, against the CHD-induced inhibition of phosphoenzyme formation from Pi. Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, sequencing, and mass spectrometry showed that Arg-198 was a single major residue protected by tight binding of F- and Mg2+ against the modification with CHD. These results indicate that modification of Arg-198 with CHD is responsible for at least a part (the portion reduced by the transition state analogs) of the CHD-induced inhibition of phosphoenzyme formation from Pi and suggest that Arg-198 is located in or close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex.
Amino Acid Sequence↗