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We report a new function for Escherichia coli DsbC, a protein best known for disulfide bond isomerization in the periplasm. We found that DsbC regulates the redox state of the single cysteine of the L-arabinose-binding protein AraF. This cysteine, which can be oxidized to a sulfenic acid, mediates the formation of a disulfide-linked homodimer under oxidative stress conditions, preventing L-arabinose binding. DsbC, unlike the homologous protein DsbG, reduces the intermolecular disulfide, restoring AraF binding properties. Thus, our results reveal a new link between oxidative protein folding and the defense mechanisms against oxidative stress.

Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.

Death receptors belong to the tumor necrosis factor receptor family. They can induce apoptosis following engagement with specific ligands and are known to play an important role in the regulation of the immune system. Here we report that epoxycyclohexenone (ECH) inhibits apoptosis induced by anti-Fas antibody, Fas ligand (FasL), or tumor necrosis factor-alpha but not by staurosporine, MG-132, C2-ceramide, or UV irradiation. These results suggest that ECH specifically blocks death receptor-mediated apoptosis. Neither the surface expression of Fas nor the Fas-FasL interaction was influenced by ECH. However, ECH did block the activation of pro-caspase-8 in the death-inducing signaling complex, although recruitment of Fas-associating death domain (FADD) and pro-caspase-8 was not affected. ECH inhibited the enzymatic activity of recombinant active caspase-8 at slightly lower concentrations than it did for active caspase-3 and active caspase-9 in vitro. However, in FasL-treated cells, ECH was only able to inhibit the activation of pro-caspase-8, and it had no effect on the already activated caspase-8 at a concentration that is effective at inhibiting Fas-induced apoptosis. ECH directly bound the large subunit of active caspase-8 that contains the active center cysteine and had a relatively higher affinity to pro-caspase-8. Moreover, compared with pro-caspase-3 and pro-caspase-9, pro-caspase-8 was predominantly depleted by biotinylated ECH with avidin beads in the cell lysates, suggesting that ECH preferentially affects pro-caspase-8. Thus, our results suggest that ECH blocks the self-activation of pro-caspase-8 in the death-inducing signaling complex and thus selectively inhibits death receptor-mediated apoptosis.

We previously designed and synthesized an NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), that showed anti-inflammatory activity in vivo. In the present study we looked into its mechanism of inhibition. DHMEQ inhibited tumor necrosis factor-alpha (TNF-alpha)- and 12-O-tetradecanoylphorbol-13-acetate-induced transcriptional activity of NF-kappaB in human T cell leukemia Jurkat cells. It also inhibited the TNF-alpha-induced DNA binding of nuclear NF-kappaB but not the phosphorylation and degradation of IkappaB. Moreover, DHMEQ inhibited the TNF-alpha-induced nuclear accumulation of p65, a component of NF-kappaB. It also inhibited TNF-alpha-induced nuclear transport of green fluorescent protein-tagged p65. On the other hand, DHMEQ did not inhibit the nuclear transport of Smad2 and large T antigen. Also, it did not inhibit TNF-alpha-induced activation of JNK but synergistically induced apoptosis with TNF-alpha in Jurkat cells. Taken together, these data indicate that DHMEQ is a unique inhibitor of NF-kappaB acting at the level of nuclear translocation.

Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, approximately 85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfoxides. An additional product with an unusual mass addition of m/z 14 (+/-0.2) was identified and corresponded to the addition of oxygen to Cys(41), conjugation to various epsilon-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCl oxidation of mutants lacking Cys(41) (Ala(41)S100A8) or specific Lys residues (e.g. Lys(34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H(2)O(2) and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products(.) In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. We previously cloned the cDNA sequence to murine mitochondrial GPAT (Yet, S-F., Lee, S., Hahm, Y. T., and Sul, H.S. (1993) Biochemistry 32, 9486-9491). We expressed the protein in insect cells which was targeted to mitochondria, purified, and reconstituted mitochondrial GPAT activity using phospholipids (Yet, S.-F., Moon, Y., and Sul, H. S. (1995) Biochemistry 34, 7303-7310). Deletion of the seven amino acids from mitochondrial GPAT, (312)IFLEGTR(318), which is highly conserved among acyltransferases in glycerolipid biosynthesis, drastically reduced mitochondrial GPAT activity. Treatment of mitochondrial GPAT with arginine-modifying agents, phenylglyoxal and cyclohexanedione, inactivated the enzyme. Two highly conserved arginine residues, Arg-318, in the seven amino stretch, and Arg-278, were identified. Substitution of Arg-318 with either alanine, histidine, or lysine reduced the mitochondrial GPAT activity by over 90%. On the other hand, although substitution of Arg-278 with alanine and histidine decreased mitochondrial GPAT activity by 90%, replacement with lysine reduced activity by only 25%. A substitution of the nonconserved Arg-279 with either alanine, histidine, or lysine did not alter mitochondrial GPAT activity. Moreover, R278K mitochondrial GPAT still showed sensitivity to arginine-modifying agents, as in the case of wild-type mitochondrial GPAT. These results suggest that Arg-318 may be critical for mitochondrial GPAT activity, whereas Arg-278 can be replaced by a basic amino acid. Examination of the other conserved residues in the seven amino acid stretch revealed that Phe-313 and Glu-315 are also important, but conservative substitutions can partially maintain activity; substitution with alanine reduced activity by 83 and 72%, respectively, whereas substituting Phe-313 with tyrosine and Glu-315 with glutamine had even lesser effect. In addition, there was no change in fatty acyl-CoA selectivity. Kinetic analysis of the R318K and R318A mitochondrial GPAT showed an 89 and 95%, respectively, decrease in catalytic efficiency but no major change in substrate binding as indicated by the K(m) values for palmitoyl-CoA and glycerol 3-phosphate. These studies indicate importance of the conserved seven amino acid stretch for mitochondrial GPAT activity and the significance of Arg-318 for catalysis.

The immunoglobulin G binding site in the globular regions of human complement subcomponent C1q has been investigated by chemical modification of histidine residues with diethylpyrocarbonate and arginine residues with phenylglyoxal and cyclohexane-1,2-dione (CHD). Only the modification of arginine residues with CHD fulfills the requirements of a specific modification without unwanted side reactions. Specific modification of arginine residues with CHD results in loss of immune complex recognition without affecting the binding of C1r2S2 to form C1. The gross structure of C1q is not changed by CHD treatment, and immune complex binding is restored to 82% of the control upon NH2OH treatment. Enzymic digestion and isolation of the modified peptides indicate that the modification by CHD of 4 to 5 arginine residues (A162, B114, B129, C156, and possibly B163) per C1q globular "head" abolishes the ability of C1q to interact with immune complexes. These residues define two areas (and possible binding sites for IgG) on the globular region of C1q: B114-B129 (site 1) and A162-(B163)-C156 (site 2). Sequence comparison and solvent exposure predictive studies favor site 2 as the immunoglobulin G binding site on the globular regions of C1q, although the participation of site 1 cannot be ruled out.

Cultured ovine tracheal epithelial cells converted arachidonic acid to prostaglandin E2 (PGE2), but microsome-containing subcellular fractions prepared from these cells under calcium-free conditions converted arachidonic acid to PGE2 and to 12-hydroxyeicosatetraenoic acid (12-HETE) at a high rate (2-4 nmol/mg of protein/15 min). Identification of the membrane-bound 12-HETE-forming activity as a 12-lipoxygenase included 12S-stereospecificity of product formation and trapping of 12-hydroperoxyeicosatetraenoic acid as a reaction product. The 12-lipoxygenase activity was extracted from cell membranes only with detergent (1% Triton X-100), and the activity (membrane-bound or detergent-solubilized) was completely inactivated by mixing with the cytosol-containing subcellular fraction. The inhibitory effect of the cytosolic fraction was reversed by treating the cytosol with GSH-depleting agents (2-cyclohexene-1-one or N-ethylmaleimide) or by mixing it with lipid hydroperoxide (13-hydroperoxyoctadecadienoic acid) at a concentration that had little direct effect on enzyme activity. Inhibition of 12-lipoxygenase activity could also be achieved by treatment of enzyme preparations with GSH at levels (0.1-10 mM) found in epithelial cell cytosol. In addition, treatment of cultured epithelial cells with a GSH-depleting agent (buthionine sulfoximine) and lipid hydroperoxide restored cellular 12-lipoxygenase activity. Little or no detectable 12-lipoxygenase activity was found in freshly isolated ovine tracheal epithelial cells, but the cytosolic 12-lipoxygenase found in freshly isolated bovine tracheal epithelial cells was relatively insensitive to regulation by GSH or lipid hydroperoxide. These observations indicate that a 12-lipoxygenase is expressed in a cryptic, microsomal-type form in primary-culture epithelial cells and that this form of the enzyme may be selectively regulated by changes in cellular oxidation-reduction conditions dependent on cytosolic levels of GSH versus lipid hydroperoxide.

The cDNA for the alpha-isoenzyme from rhesus monkey (Macaca mulatta) liver was cloned and expressed in yeast. The alpha-isoenzymes of human and monkey liver alcohol dehydrogenase differ from the other human and horse liver enzymes in having Met57, Ala93, and Val116 instead of Leu57, Phe93, and Leu116 in the substrate binding pocket and Gly47 instead of Arg47 near the pyrophosphate moiety of the coenzyme. The effects of these differences on the kinetic mechanism, substrate specificity, and coenzyme binding were studied with the purified, recombinant monkey alpha-isoenzyme (MmADH alpha) and mutated enzymes with Gly47 substituted with His or Arg. The mechanism appears to be random for the binding of NAD+ and ethanol and ordered for NADH and acetaldehyde, with formation of a dead-end enzyme-NADH-ethanol complex. MmADH alpha reacts 130-fold slower (V/K) with ethanol and 3-25-fold slower with 2-methyl alcohols but 20-fold faster with cyclohexanol, as compared with horse (Equus caballus) liver EE isoenzyme (EqADH). MmADH alpha is stereoselective for the R isomer of 2-butanol, whereas EqADH favors the S isomer. Both enzymes have comparable reactivity with larger primary alcohols. MmADH alpha is more reactive with secondary alcohols and has highest activity with cyclohexanol. However, it does not react with steroids such as 5 beta-androstane-17 beta-ol-3-one. Molecular modeling suggests that the differences between MmADH alpha and EqADH are a result of the substitution of Ala for Phe93 and Thr for Ser48. MmADH alpha binds NAD+ most rapidly when a group with a pK of 7.4 is unprotonated, implicating His51 in this reaction. The G47R substitution decreased the dissociation constants for NAD+ and NADH and turnover numbers only about 2-fold, whereas the G47H substitution increased dissociation constants 7-14-fold and turnover numbers 4-fold. A basic residue at position 47 is not crucial for activity, as multiple interactions determine coenzyme affinity.

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.

Serratia marcescens anthranilate synthase Component I (AS I) was purified from a plasmid-containing Escherichia coli strain. Residues essential for AS I function were studied by chemical modification reactions. Phenylglyoxal and 1,2-cyclohexanedione modified 2-5 arginine residues and inactivated AS I. The substrate chorismate reduced the rate of inactivation. Analysis of inactivation data indicated that 1 arginine residue is essential for activity. Histidine residues in AS I were modified by ethoxyformic anhydride and by photooxidation. Enzyme inactivation accompanied modification of histidine residues. Inactivation was prevented by substrate. Comparison of the number of carbethoxy groups incorporated between substrate-protected and unprotected AS I indicated that 1 histidine residue is required for activity. AS I was also inactivated by bromopyruvate. Substrate retarded inactivation by bromopyruvate. A differential labeling experiment indicated that the loss of AS I activity was correlated with alkylation of 1 cysteine residue. A tryptic peptide containing the essential cysteine residue was isolated. The peptide has the amino acid sequence of Ile-Cys-Gln-Ala-Gly-Ser-Arg.

In this study we examine the effects of intravenous ethyl oleate emulsions on the metabolism of native and cyclohexanedione-modified human low density lipoprotein in rabbits. Treatment produced a highly significant fall in receptor-independent catabolism as measured by the fractional clearance rate of cyclohexanedione-modified low density lipoprotein. Receptor-dependent catabolism (the difference between the fractional clearance rates of native and cyclohexanedione-modified low density lipoprotein) was variably affected with some animals showing a decrease in receptor activity. These data suggest that the reticuloendothelial system makes a substantial contribution to receptor-independent low density lipoprotein catabolism in the rabbit.

Incubation of homogeneous preparations of L-threonine dehydrogenase from Escherichia coli with 2,3-butanedione, 2,3-pentanedione, phenylglyoxal, or 1,2-cyclohexanedione causes a time- and concentration-dependent loss of enzymatic activity; plots of log percent activity remaining versus time are linear to greater than 90% inactivation, indicative of pseudo-first order inactivation kinetics. The reaction order with respect to the concentration of modifying reagent is approximately 1.0 in each case suggesting that the loss of catalytic activity is due to one molecule of modifier reacting with each active unit of enzyme. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the dehydrogenase. Essentially the same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. NADH (25 mM) and NAD+ (70 mM) give full protection against inactivation whereas much higher concentrations (i.e. 150 mM) of L-threonine or L-threonine amide provide a maximum of 80-85% protection. Amino acid analyses coupled with quantitative sulfhydryl group determinations show that enzyme inactivated 95% by 2,3-butanedione loses 7.5 arginine residues (out of 16 total)/enzyme subunit with no significant change in other amino acid residues. In contrast, only 2.4 arginine residues/subunit are modified in the presence of 80 mM NAD+. Analysis of the course of modification and inactivation by the statistical method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558) demonstrates that inactivation of threonine dehydrogenase correlates with the loss of 1 "essential" arginine residue/subunit which quite likely is located in the NAD+/NADH binding site.

This work presents strong evidence that the role of the active site arginine in D-amino acid oxidase is to act as a positively charged group interacting with the flavin N(1)-C(2) = 0 locus. Modification with cyclohexanedione, which has been shown previously to modify specifically an active site arginine in D-amino acid oxidase (Ferti, C., Curti, B., Simonetta, M. P., Ronchi, S., Galliano, M., and Minchiotti, L. (1981) Eur. J. Biochem. 119, 553-557) destroys the ability of D-amino acid oxidase to stabilize the benzoquinoid type spectrum of 8-mercapto-FAD and destroys the ability to form a flavin N-5 adduct with sulfite. Both of these properties have been attributed to the presence of such a group. The active site lysine, histidine, and tyrosine have been ruled out as possibilities for such a group. In addition, the reactivity of flavoproteins containing 8-mercaptoflavin with sulfite has been examined and falls into the same two general classes as the reactivity of the native flavoproteins: oxidases form N-5 adducts while all of the other 8-mercaptoflavoproteins examined do not, forming instead the 8-sulfonate flavin.

When mouse pancreatic "minilobules" prelabeled with either [14C]arachidonic acid (AA), [14C]stearic acid (SA), or [3H]glycerol were stimulated with the secretogogue, caerulein, there was a 60-70% loss in radioactivity in phosphatidylinositol (PI) at 30 min. This loss was accompanied by the formation of [14C] phosphatidic acid (PA), [14C]diacylglycerol (DG), [14C] triacylglycerol (TG), and free [14C]AA, [14C]SA, and [3H]glycerol. The loss in radioactive PI was the same as the loss in chemically measured PI-phosphorus. Thirty to fifty per cent of the caerulein-induced loss of prelabeled PI could be accounted for as free [14C]AA, [14C]SA, or [3H]glycerol. Increased incorporation of fatty acid or glycerol residues into DG, PA, and TG accounted for the balance of the loss in PI. The specific DG-lipase inhibitor, RHC 80267, markedly inhibited the caerulein-stimulated release of [14C]AA, [14C]SA, and [3H]glycerol and roughly doubled the caerulein-induced increment in [14C]AA-, [14C]SA-, or [3H]glycerol-labeled DG, showing that the source of the caerulein-induced increment in fatty acids and glycerol was DG. When the PI was prelabeled with either [32P] orthophosphate, [3H]myoinositol, or [3H]glycerol, only 1% or less of the radioactivity in PI was in lysophosphatidylinositol (LPI), and there was no increase in radioactivity in LPI on stimulation with caerulein. These observations, taken together, argue strongly for a phospholipase C-catalyzed breakdown of PI followed by DG-lipase and argue against any significant involvement of phospholipase A2 in PI degradation in mouse pancreas. The formation of substantial amounts of free [14C]AA on stimulation supports the view that, among other things, the phosphoinositide effect in the exocrine pancreas serves to generate arachidonate (and its metabolites). The release of appreciable amounts of free fatty acids and glycerol shows that a significant portion of the DG formed as a result of caerulein-stimulated PI breakdown is not conserved in the phosphoinositide cycle.

The metabolism of exogenous [3H]diacylglycerols by intact human platelets was studied in order to examine: the metabolic fate of these second messengers in an intact cell, the effect of diacylglycerol kinase and diacylglycerol lipase inhibitors on this metabolism, the effect of agonist stimulation on metabolism, and the dependence of metabolism on diacylglycerol chain length. When 2.5 microM [3H]dioctanoylglycerol (diC8) was added to 10(9) platelets it was rapidly metabolized; 80% was converted to various products in 2.5 min. Initially, 40% was recovered as 3H-labeled phospholipid (predominantly phosphatidic acid) reflecting the action of diacylglycerol kinase, 20% was recovered as [3H]glycerol due to the action of diacylglycerol and monoacylglycerol lipases, and small amounts were recovered as triacylglycerol and monoacylglycerol. Thrombin stimulation of platelets did not affect the rate or pathway of metabolism. Pretreatment of platelets with the diacylglycerol kinase inhibitors, diC8ethyleneglycol or 1-monooleoylglycerol, inhibited 3H-labeled phospholipid production 47% and 75%, respectively, and resulted in a longer lived diC8 signal. The diacylglycerol lipase inhibitor, RHC 80267, inhibited the production of water-soluble metabolites 75%. Despite inhibition of the lipase, the overall metabolism of exogenous [3H]diC8 occurred at a similar rate as in control platelets due to an increased flux towards phospholipid. The ability of exogenous diacylglycerols to be metabolized by diacylglycerol kinase correlated well with their ability to activate protein kinase C in platelets. [3H]Dibutyroylglycerol, didodecanoylglycerol, and ditetradecanoylglycerol, were not metabolized by this route. These diacylglycerols were still metabolized via the lipase pathway. The results indicate that platelets possess potent attenuation systems to defend against the accumulation of diacylglycerol second messengers, and that the primary metabolic fate of cell-permeable, exogenous diacylglycerols is conversion to phosphatidic acid.

We have chemically modified groups of amino acids in the sequence of alpha-cobratoxin and have studied the derivatives as to their affinity of binding to the acetylcholine receptor protein from Torpedo marmorata. (i) The toxin derivatives which were fully modified at lysine (penta-epsilon-N,N-dimethyl lysine; penta-epsilon-N-acetyl lysine), arginine (penta-N7,N8-(1,2-dihydroxycyclohex-1,2-ylene arginine), and tyrosine (mononitrotyrosine) all had significant remaining toxicity and affinity of binding. (ii) The "extra" disulfide of alpha-cobratoxin was selectively reduced and alkylated. Depending on the charge, size, and hydrophobicity of the attached groups, derivatives were obtained that bound to the acetylcholine receptor with higher (di-S-carboxyamidomethyl), about equal (di-S-pyridylethyl), or lower (di-iodoacetaminoethylnaphthylamine-5-sulfonic acid) affinity than the unmodified toxin. (iii) A fully reduced and carbamidomethylated derivative of alpha-cobratoxin obtained by repeating the procedure for selective reduction six times still bound with appreciable affinity (KD approximately 3 X 10(-6) M) to the acetylcholine receptor. We conclude that neither a single positively charged residue nor tyrosine nor the integrity of the disulfides is absolutely essential for toxicity. Furthermore, the single tyrosine and the area around the extra disulfide do not participate in the binding to the receptor. Together with previous findings on this interaction, this suggests a multipoint attachment of toxin and receptor involving several locally separate structural elements of the toxin.

Human serum albumin (HSA) has been chemically modified with 1,2-cyclohexanedione and N-acetylimidazole under nondenaturing conditions. Derivatives, in which 10 arginine residues (1,2-dihydroxycyclohex-1,2-ylene (DHCH)-HSA), 56 to 57 lysine and 5 tyrosine residues (acetyl-HSA), or 56 to 57 lysine residues alone (O-deacetyl-HSA) are modified, have been isolated. Their conformation has been tested by circular dichroism measurement, gel filtration on Sephadex G-150, and ultracentrifugation. From these analyses and binding studies it is concluded that only insignificant changes of conformation have occurred. The binding properties of the HSA derivatives have been tested with bilirubin, diazepam (a benzodiazepine drug), phenylbutazone, and indomethacin by circular dichroism. The binding of diazepam to DHCH-HSA is almost completely inhibited and that of phenylbutazone and indomethacin decreased, while the binding of bilirubin is essentially unaffected. In acetyl-HSA and O-deacetyl-HSA, the bilirubin binding is significantly decreased, while the binding of the drugs mentioned is less affected. It is concluded that bilirubin and the drugs bind to two separate sites which contain positive charges essential for the binding properties. The charge(s) in the benzodiazepine site from arginine.

Receptor-estradiol complexes (RE2) formed at 0 degree C in hypotonic buffers bind poorly to nuclei (nonactivated state); their sedimentation coefficient in low or high salt sucrose density gradients (SDG) is 8 S or 4 S, respectively (untransformed state); estradiol dissociates from untransformed RE2 at a high rate (k-1 = 0.44 min-1). Brief heating (28 degrees C, 30 min) induces activation (increased binding of RE2 to nuclei and polyanions), transformation (formation of receptor dimers which sediment at 6 S in 0.4 M KCl/borate SDG) and RE2 transition into a state from which E2 dissociates at a lower rate (k-2 = 8 X 10(-3) min-1). We have examined the role of arginyl residues in the above changes in receptor properties. It is well established (Patthy, L., and Smith, E. L. (1975) J. Biol. Chem. 250, 557-564; 565-569) that 1,2-cyclohexanedione (1,2-CHD) is a highly specific arginine-modifying agent; in borate buffer at 28 degrees C, but not at 0 degrees C, peptide arginyls are covalently modified. RE2 complexes heated in the presence of 1,2-CHD (50 mM) bind poorly to nuclei; 1,4-cyclohexamedione and 1,2-cyclohexanediol had no effect. This reagent also prevents the temperature-induced transition of RE2 into a state with slow E2 dissociation rates although it does not interfere with heat transformation (formation of 6 S dimer). Modification of heat-activated and transformed RE2 by 1,2-CHD causes a loss in receptor binding to nuclei and alters RE2 from a state with slow into a state with fast E2 dissociation rates, although the receptor remains unaltered in the transformed 6 S state. At 0 degree C, i.e. in the absence of covalent arginyl modification, 1,2-CHD promotes dissociation of the 8 S aggregate into 4.6 S subunits which bind to nuclei to the same extent as heat-transformed control RE2. Heating of the molybdate-stabilized 8 S receptor in the presence of 1,2-CHD yields a nonactivated 8 S receptor (4.6 S on high salt SDG); removal of molybdate and unreacted 1,2-CHD by gel filtration at 0 degree C followed by exposure to high ionic strength causes 8 S to 4 S dissociation; these 4 S subunits, however, do not bind to nuclei, suggesting that their nucleotropic domain was accessible to 1,2-CHD modification while the receptor was in the aggregated 8 S state. It is proposed that the nuclear binding site of the estrogen receptor contains arginyl residues. Furthermore, a distinct set of arginyl residues appears to be related to the estrogen-binding domain; its integrity is required for the heat-induced formation and maintenance of the RE2 state with slow E2 dissociation.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibody BBM.1 (Brodsky, F.M., Bodmer, W. F., and Parham, P. (1979) Eur. J. Immunol. 9, 536-545) identifies an antigenic determinant of human beta 2-microglobulin (beta 2-M). The antibody binds free and HLA-A,B-associated beta 2-M with similar affinity, showing that the BBM.1 antigenic determinant does not involve residues of beta 2-M that interact with HLA-A,B heavy chains. Peptides (SWH.1-5) synthesized from residues 35-50 of the beta 2-M sequence specifically inhibit the binding of BBM.1 to cell surfaces. Their inhibitory activity is destroyed by trypsin treatment. The observations (i) that BBM.1 does not bind to beta 2-M of species other than man, gorilla, and chimpanzee and (ii) that arginine 45 is the only human-specific residue between positions 35 and 50 suggested that this residue might be part of the BBM.1 antigenic determinant. This hypothesis was confirmed by reversible modification of arginine residues with cyclohexanedione. Modification of arginines in native beta 2-M and of the single arginine, corresponding to position 45, in the peptide SWH.5 resulted in up to 95% loss of BBM.1 inhibitory activity. Reversal of the modification by treatment with hydroxylamine resulted in complete recovery of activity. Rabbit antibodies elicited by immunization of SWH.5 conjugated to bovine serum albumin showed no detectable reaction with native beta 2-M but did specifically react with human beta 2-M after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoresis onto nitrocellulose. These results thus identify a region around residue 45 of the beta 2-M polypeptide which is exposed to the environment and not involved in binding HLA-A,B heavy chain. Analysis of the beta 2-M sequence by calculating local hydrophilicity indices (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828) agree with this region being a major antigenic determinant. Models of beta 2-M structure as an immunoglobulin domain show this region of polypeptide to be part of a loop between the two layers of beta-pleated sheet, also consistent with it being a major antigenic determinant. The position of the loop favors a model in which beta 2-M interacts with HLA across the four-stranded beta-pleated sheet like an immunoglobulin constant region domain.

UDP-glucose 4-epimerase from Saccharomyces fragilis was inactivated by the arginine-specific reagents phenylglyoxal, 1,2-cyclohexanedione, and 2,3-butanedione following pseudo first order reaction kinetics. The reaction order with respect to phenylglyoxal was 1.8 and that with respect to the other two diones was close to unity. Protection afforded by substrate and competitive inhibitors against inactivation by phenylglyoxal and the reduced interaction of 1-anilinonaphthalene 8-sulfonic acid, a fluorescent probe for the substrate-binding region after phenylglyoxal modification, suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of UMP, a ligand known to interact at the substrate-binding region, showed that only the arginine residue at the active site could be modified by phenylglyoxal. The characteristic coenzyme fluorescence of the yeast enzyme was found to be enhanced three times in phenylglyoxal-inactivated enzyme suggesting the incorporation of the phenyl ring near the pyridine moiety of NAD.

The chloroperoxidase-peracetic acid-chloride-2-chlorodimedone system is used as a model for the investigation of enzymatic halogenation reactions. Systematic variation of the concentrations of the three substrates under steady state conditions yields sets of kinetic parameters containing both kinetic and mechanistic information. Three distinct enzyme species are involved in the halogenation cycle: native enzyme, compound I, and a short-lived halogenating intermediate. Analysis of the kinetic data is complicated by the fact that chloride serves as a substrate in the second step and as an inhibitor of the first step of the overall halogenation reaction. The inhibitor binding site on the native enzyme must be protonated prior to the binding of chloride. Chloride appears to be a competitive inhibitor for both compound I formation and cyanide binding to chloroperoxidase. Only the latter reaction can be studied directly in stopped-flow experiments since compound I disappears rapidly by reacting with chloride present in solution. Rate constants are calculated for the individual steps of the reaction at four different pH values. The rate constant for compound I formation is independent of pH, but the actual rate is reduced at lower pH values due to pH dependence of the chloride inhibition. The rate constant for the oxidation of chloride by compound I decreases with increasing pH. The rate of the halogenation step appears to be independent of pH.

Chemical modification of 2 +/- 0.5 arginine residues of porcine pancreatic elastase by 1,2-cyclohexanedione leads to an 85 +/- 5% loss of activity with the specific substrate N-succinyltrialanine p-nitroanilide. Modification of additional arginines does not completely abolish the enzyme activity. The modification reaction is very fast (second order rate constant = 0.24 M-1 S-1) and involves only arginine residues. Acetyltetraalanine or trifluoroacetyltetraalanine decreases the rate of cyclohexanedione-induced inactivation of the enzyme but does not significantly change the number of modified arginine residues. Other dicarbonyl reagents, butanedione or phenylglyoxal, also react with elastase but at much lower rates. Cyclohexanedione-modified elastase is partially active against a series of synthetic substrates of varying chain length. The partial inhibition results from a 2- to 5-fold increase in Km while kappa cat is increased for most substrates. For N-succinyltrialanine p-nitroanilide both the acylation and deacylation rate constants are decreased. The Ki values of a series of acetylated and trifluoroacetylated inhibitors increase 2- to 5-fold. Modified elastase is still able to react with fibrous elastin and with plasma alpha 1-proteinase inhibitor but at significantly lower rates. Modification of one arginine residue alters the properties of the calcium-binding site of elastase as demonstrated by terbium luminescence experiments. The affinity of enzyme for terbium is decreased by a factor of 10 and the circularly polarized luminescence spectrum of the terbium-elastase complex is considerably flattened. Modification of further arginine residues does not increase the extent of these alterations. Circular dichroism shows that the overall conformation of elastase is not altered following arginine modification. We speculate that the two residues modified by cyclohexanedione are Arg 65, located at about 8 A from the metal ion-binding site, and Arg 217A, located at the S'3 subsite of elastase.

This study examines the effects of cholestyramine (2 g/day) on the plasma clearance and tissue uptake of human low density lipoprotein (LDL) in rabbits. 1,2-Cyclohexanedione modification of human LDL abolishes its recognition by high affinity cell membrane receptors in vitro and delays its plasma clearance in comparison to native LDL. Consequently, the difference between the fractional rates of catabolism of simultaneously injected native and cyclohexanedione-treated LDL is an index of in vivo receptor-mediated clearance of the lipoprotein. When human 125I-LDL and 131I-cyclohexanedione-treated LDL were injected into rabbits, 44% of the lipoprotein was cleared from the plasma by the receptor mechanism. Various tissues were removed from the animals at the end of the turnover study and their relative uptakes of 125I native and 131I-cyclohexanedione-treated LDL were measured. All exhibited receptor activity to some extent, incorporating more native than cyclohexanedione-modified LDL. The greatest receptor activity per g of tissue was found in lymph nodes, spleen, and liver and, in terms of whole organ uptake, the liver played a major role in LDL catabolism. Treatment of the rabbits with cholestyramine lowered the circulating LDL cholesterol level by promoting its clearance (120%, p < 0.001) via the receptor pathway. This was associated with a virtual doubling of receptor-mediated incorporation of the lipoprotein into the liver. These results suggest that the drain which cholestyramine induces in the hepatic cholesterol pool promotes LDL receptor activity in this organ and thereby lowers the level of circulating LDL.

Treatment of homogeneous preparations of Escherichia coli 2-keto-4-hydroxyglutarate aldolase with 1,2-cyclohexanedione, 2,3-butanedione, phenylglyoxal, or 2,4-pentanedione results in a time- and concentration-dependent loss of enzymatic activity; the kinetics of inactivation are pseudo-first order. Cyclohexanedione is the most effective modifier; a plot of log (1000/t 1/2) versus log [cyclohexanedione] gives a straight line with slope = 1.1, indicating that one molecule of modifier reacts with each active unit of enzyme. The kinetics of inactivation are first order with respect to cyclohexanedione, suggesting that the loss of activity is due to modification of 1 arginine residue/subunit. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced structural alteration of the aldolase. The same Km but decreased Vmax values are obtained when partially inactivated enzyme is compared with native. Amino acid analyses of 95% inactivated aldolase show the loss of 1 arginine/subunit with no significant change in other amino acid residues. Considerable protection against inactivation is provided by the substrates 2-keto-4-hydroxyglutarate and pyruvate (75 and 50%, respectively) and to a lesser extent (40 and 35%, respectively) by analogs like 2-keto-4-hydroxybutyrate and 2-keto-3-deoxyarabonate. In contrast, formaldehyde or glycolaldehyde (analogs of glyoxylate) under similar conditions show no protective effect. These results indicate that an arginine residue is required for E. coli 2-keto-4-hydroxyglutarate aldolase activity; it most likely participates in the active site of the enzyme by interacting with the carboxylate anion of the pyruvate-forming moiety of 2-keto-4-hydroxyglutarate.

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of catecholamines was mediated by a beta-receptor interaction and cAMP (Abumrad, N.A., Park, C.R., and Whitesell, R. R. (1986) J. Biol. Chem. 261, 13082-13086). In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the cAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phosphodiesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 microM) of the phosphodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 microgram/ml) gradually abolished insulin's antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 microM methylisobutylxanthine. Also consistent with involvement of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin's responsiveness. Treatment of cells with adenosine deaminase (1 unit/ml) enhanced the effect of norepinephrine by about 30%. A 10-fold higher range of insulin concentrations was then required to produce inhibition of fatty acid transport. The effect of adenosine removal was reversed by addition of phenylisopropyladenosine (500 nM), which is resistant to hydrolysis by the deaminase. Finally, exposure of insulin-treated cells (1 nM for 5 min) to dinitrophenol (1 mM for 5 min) reversed insulin action, consistent with reports of reversal of insulin's activation of the phosphodiesterase. In conclusion, our studies support the involvement of cAMP lowering in insulin's antagonism of fatty acid transport stimulation in the adipocyte.

Reaction of phenylglyoxal, a reagent specific for arginine residues, with porcine phospholipiase A2 results in complete elimination of catalytic activity. The modification reaction is markedly dependent on pH. Other dicarbonyl compounds such as 2,3-butanedione and 1,2-cyclohexanedione also react with the enzyme to cause loss of activity but at significantly slower rates. At pH 7.0, the inactivation can be slightly retarded in the presence of Ca2+ and almost completely prevented in the presence of n-alkylphosphorylchline inhibitors. At pH 8.5, the n-alkylphosphorylcholine inhibitors are less effective. The decrease in enzymatic activity correlates with the modification of about 1 arginine residue/phospholipase A2 molecule. The data suggest that the arginine is involved in the binding of the phosphate portion of the substrate to the enzyme.

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.

Human glucose phosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. A specific lysine was found to stoichiometrically react with pyridoxal 5'-phosphate forming a reversible Schiff base which could be reduced with NaBH4. The covalently modified enzyme was specifically cleaved with hydroxylamine at three labile Asn-Gly sequences yielding a series of peptides which were separated by sodium dodecyl sulfate-polyacrylamide electrophoresis. The modified lysine was located in the COOH-terminal peptide. A critical arginine residue/subunit was found to be stoichiometrically modified with either 2,3-butadione or cyclohexadione. At high concentrations of butadione, an irreversible nonspecific modification of essentially all arginines occurred. An essential tryptophan residue was found to be stoichiometrically modified with N-bromosuccinimide in a similar fashion. Each of the chemical modifications of these three residues followed pseudo-first order and rate saturation kinetics and the modifications were prevented by the presence of substrates or competitive inhibitors. Circular dichroic spectral studies and analytical gel filtration indicated that these modifications have no effect on the quarternary structure and little effect on the secondary and tertiary structures of the enzyme. However, the extensive modification of arginine with butadione caused a dissociation of the enzyme into monomers and significant changes in tertiary structure. These studies provide new insights into functional aspects of isomerization and also provide an effective method for evaluating structural consequences of chemical or genetic modification of the enzyme.

The presence of arginine at the active site of avian liver phosphoenolpyruvate carboxykinase was studied by chemical modification followed by a characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione all irreversibly inhibit the enzyme with second-order rate constants of 3.42 M-1 min-1, 3.13 M-1 min-1 and 0.313 M-1 min-1, respectively. The substrates phosphoenolpyruvate, IDP, and the activator Mn2+ offer little to modest protection from inhibition. Either CO2 or CO2 in the presence of any of the other substrates elicited potent protection against modification. Protection by CO2 against modification by phenylglyoxal or 1,2-cyclohexanedione gave a biphasic pattern. Rapid loss in activity to 40-60% occurred, followed by a very slow loss. Kinetics of inhibition suggest that the modification of arginine is specific and leads to loss of enzymatic activity. Substrate protection studies indicate an arginine residue(s) at the CO2 site of phosphoenolpyruvate carboxykinase. Apparently no arginine residues are at the binding site of the phosphate-containing substrates. Partially inactive (40-60% activity) enzyme, formed in the presence of CO2, has a slight change of its kinetic constants, and no alteration of its binding parameters or secondary structure as demonstrated by kinetic, proton relaxation rate, and circular dichroism studies. Labeling of enzyme with [(7-)14C]phenylglyoxal in the presence of CO2 (40-60% activity) showed 2 mol of phenylglyoxal/enzyme or 1 arginine or cysteine residue modified. Labeling of phosphoenolpyruvate carboxykinase in the absence of CO2 yielded 6 mol of label/enzyme. Labeling results indicate that avian phosphoenolpyruvate carboxykinase has 2 or 3 reactive arginine residues out of a total of 52 and only 1 or 2 are located at the active site and are involved in CO2 binding and activation.

The role of diacylglycerol (DG) as a source of arachidonic acid during gonadotropin-releasing hormone (GnRH) stimulation of gonadotropin secretion was analyzed in primary cultures of rat anterior pituitary cells. An inhibitor of DG lipase (RHC 80267, RHC) caused dose-dependent blockade of GnRH-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. The DG lipase inhibitor did not alter gonadotropin responses to arachidonic acid, and addition of arachidonic acid reversed its inhibition of GnRH-stimulated LH and FSH release. In [3H]arachidonic acid-prelabeled cells, incubation with RHC increased the accumulation of [3H]DG. These results suggest that DG lipase participates in GnRH action and that arachidonic acid mobilization from DG is involved in the mechanism of gonadotropin release. Gonadotropin responses to tetradecanoyl phorbol acetate and dioctanoyl glycerol were not altered by RHC, and the addition of these activators of protein kinase C (Ca2+- and phospholipid-dependent enzyme) did not prevent the inhibition of GnRH-induced gonadotropin release by RHC. Activation of phospholipase A2 by melittin increased LH and FSH secretion, whereas blockade of this enzyme by quinacrine reduced GnRH-stimulated hormone release. However, RHC did not diminish the gonadotropin response to melittin. The inhibitory actions of RHC and quinacrine were additive and were reversed by concomitant treatment with arachidonic acid. Ionomycin also increased LH and FSH release, and the gonadotropin responses to the ionophore were unaltered by RHC but were reduced by quinacrine. Incubation of cells in Ca2+-depleted (+/- [ethylenebis(oxyethylenenitrilo)]tetraacetic acid) medium reduced but did not abolish the LH and FSH releasing activity of GnRH. Treatment with RHC also reduced the gonadotropin responses to GnRH under Ca2+-depleted conditions. These observations indicate that RHC inhibition of GnRH action is not due to nonspecific actions on Ca2+ entry, protein kinase C activation and actions, nor phospholipase A2 enzyme activity. The results of this study provide further evidence for an extracellular Ca2+-independent mechanism of GnRH action, and suggest that GnRH causes mobilization of arachidonic acid by two distinct lipases, namely, phospholipase A2 and DG lipase, during stimulation of gonadotropin secretion.