Analysis of sulfate in complex carbohydrates.
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Carbohydrates↗
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Determination of peroxidative halogenation in mixtures of chloride and bromide.
A method for the differentiation of chlorinated and brominated products from peroxidative oxidation of mixtures of the halides is presented. Chlorination or bromination of monochlorodimedone (MCD) by fungal chloroperoxidase (CPO) was measured by loss of MCD absorbance. Although the Vmax was similar for both halides [approximately 0.08 mM (2 min)-1], the apparent Km for chlorination was 10 times greater than that for bromination (5.88 vs 0.67 mM). Chlorination was also quantitated as I3- produced from N-chlorotaurine and I-. The Vmax [0.076 mM (2 min)-1] and apparent Km (6.31 mM) determined by this method agreed with those determined with MCD. Selective reduction by H2O2 of the I-oxidizing potential of N-bromotaurine allowed determination of the brominated product from the difference between the amounts of halogenated MCD and N-chlorotaurine. The brominated product predominated at saturating and at physiologic halide levels. Hence, it is suggested that Br- plays a significant role in halogenation even though in vivo levels of Cl- are equal to or greater than 1000 times those Br-.
Bromides↗
The measurement of negative charge content in cartilage using a colloid titration technique.
A colloid titration technique has been used to determine the sulfate and carboxylate content of various glycosaminoglycans and has been validated by comparing the results with data obtained using well-established techniques. The method has been applied to the measurement of the negative charge content of cartilage slices at various depths from the articular surface and to the determination of sulfate and carboxylate contents in bovine nasal septa. Titrations of nasal septa were performed on milled cartilage, on cartilage digested with papain and on proteoglycans purified by cesium chloride gradient centrifugation of guanidinium chloride extracts. The sulfate content was similar for all three preparations (0.5 mu eq per milligram dry cartilage). However, the carboxylate content determined on milled cartilage was 40% higher than that obtained for cartilage digested with papain or for purified proteoglycans; this implies the possible contribution of carboxyl groups from structural glycoproteins present in the extracellular matrix. The carboxylate content determined on purified proteoglycans was in excellent agreement with values calculated from chemical analyses.
Animals↗
Role of arginine residues in ovine lutropin: reversible modification by 1,2-cyclohexanedione.
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Animals↗
Evidence for an essential role for arginyl residues for yeast phosphoglycerate kinase.
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Adenosine Triphosphate↗
Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.
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3-Hydroxysteroid Dehydrogenases↗
Evidence for a radical mechanism of halogenation of monochlorodimedone catalyzed by chloroperoxidase.
A radical species of monochlorodimedone has been characterized by its high reactivity with molecular O2. Horseradish peroxidase greatly accelerated O2 uptake by acidic solutions of this substrate; the enzymatic reaction required exogenous H2O2 only with freshly prepared substrate solutions, and the total substrate oxidized was equal to the sum of H2O2 added and O2 consumed. However, with excess Br- and horseradish peroxidase, or high Br- or Cl- and chloroperoxidase, a 1:1 stoichiometry between H2O2 and substrate was observed. In the absence of halide, the stoichiometry of the chloroperoxidase-catalyzed oxidation of monochlorodimedone changed to two molecules of the organic donor per H2O2. Moreover, in the absence of halide, at substrate:H2O2 ratios greater than 2.0, chloroperoxidase catalyzed significant O2 uptake; this enzyme-dependent autoxidation of monochlorodimedone also occurred in the presence of Cl- or Br-, when H2O2 was limiting. These data, and recent evidence from this laboratory for free hypohalous acid as the first product of chloroperoxidase-catalyzed halide oxidation [B. W. Griffin (1983) Biochem. Biophys. Res. Commun. 116, 873-879], strongly support a mixed enzymatic/nonenzymatic radical chain process as the mechanism for halogenation of monochlorodimedone by chloroperoxidase. Both horseradish peroxidase and chloroperoxidase can catalyze either bromination or oxidation of this substrate, depending on the experimental conditions. Implications of these results for the mechanism of HOCl formation catalyzed by chloroperoxidase are considered.
Chloride Peroxidase↗
On the mechanism of chlorination by chloroperoxidase.
Spectral-scan results obtained on the millisecond time scale are reported for reactions of chloroperoxidase with peracetic acid and chloride ion in both the presence and the absence of monochlorodimedone. A multimixing experiment is performed in which stoichiometric amounts of chloroperoxidase and peracetic acid are premixed for 0.7 s before the resultant compound I is reacted with chloride ion. The combined results show that the only detectable enzyme intermediate species is compound I (except in very late stages of the reaction), that the disappearance of compound I is accelerated by the presence of chloride ion, and that it is further accelerated if both chloride and monochlorodimedone are present. It is concluded that compound I is an obligate intermediate species in the reaction. Experiments are performed on the reaction of monochlorodimedone with hypochlorous acid in both the presence and the absence of added chloride ion, but in the absence of chloroperoxidase. The presence of chloride ion greatly accelerates the reaction rate apparently by setting off a chlorine chain reaction. This reaction would be important in the enzyme-catalyzed reaction if hypochlorous acid were liberated into the solution. A careful analysis of steady-state kinetic results shows that in the chlorination of monochlorodimedone at least, liberation of free hypochlorous acid is not important in the enzyme-catalyzed pathway. Rather the reaction proceeds from compound I to formation of iron(III)-OCl by chloride ion addition to the ferryl oxygen atom. This obligate intermediate species then chlorinates the substrate. It is well described as enzyme-activated hypochlorous acid, in which replacement of the proton in HOCl by the heme iron ion produces a Cl+ species of great potency. Thus the enzyme controls chlorination of monochlorodimedone rather than unleashing an uncontrolled chain reaction in which it would be rapidly destroyed.
Chemical Phenomena↗
Inhibitors of diacylglycerol lipase and diacylglycerol kinase inhibit carbamylcholine-stimulated responses in guinea pig pancreatic minilobules.
We earlier showed that the diacylglycerol (DG) lipase inhibitor, RHC 80267, increased the steady-state level of DG and inhibited the release of arachidonic acid (AA) in carbamylcholine (CCh)-stimulated pancreatic minilobules (J. F. Dixon and L. E. Hokin, (1984) J. Biol. Chem. 259, 14418-14425). There was no effect on phospholipid metabolism. We have now investigated the effect of RHC 80267 on CCh-stimulated formation of inositol monophosphate formation, cGMP formation, and amylase release. CCh (10 microM) increased cGMP formation by approximately 20-fold, and this response was inhibited 55-75% by RHC 80267 (75-100 microM). RHC 80267 had no effect on either nitroprusside- or calcium ionophore-stimulated cGMP formation, arguing against a direct inhibition of guanylate cyclase by RHC 80267. Arachidonic acid, the release of which is inhibited by RHC 80267, neither stimulated cGMP formation nor reversed the effect of RHC 80267 on CCh-stimulated cGMP formation. This suggests, but does not prove, that the rise in cGMP in response to CCh is not due to an increase in AA as has been suggested. Both phorbol myristate acetate (25 nM) and the DG kinase inhibitor R 59022 (10 microM) inhibited CCh-stimulated cGMP formation by 40%. RHC 80267 also inhibited CCh-stimulated inositol phosphate accumulation and amylase release by 60 and 40%, respectively. The data suggest that the inhibition of CCh-stimulated cGMP formation and other muscarinic responses by RHC 80267 is probably the result of feedback inhibition of the cholinergic receptor via activation of protein kinase C by the elevated DG.
Amylases↗
Phosphorylation of glucokinase from rat liver in vitro by protein kinase A with a concomitant decrease of its activity.
Glucokinase, purified from rat liver, was phosphorylated to an extent of 1 mol [32P]-phosphate/mol of enzyme when incubated with [32P]ATP and protein kinase A from pig or rabbit muscle. The phosphate was bound to serine residues. K0.5 increased and Vmax decreased upon phosphorylation. The phosphate group was removed during incubation of the phosphorylated glucokinase with alkaline phosphatase. Enzymatically inactive glucokinase was not phosphorylated by the protein kinase.
Animals↗
Modification of arginine residues in human growth hormone by 1,2-cyclohexanedione: effects on the binding capacity to lactogenic and somatogenic receptors.
Reactivity of arginine residues in human growth hormone was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit to a pseudo first order curve, with an apparent velocity constant k = 1.26 x 10(-2) min-1 and a maximum modification of 9.6 out of the 11 arginines of the molecule. Modification led to a decrease in binding capacity to both lactogenic and somatogenic rat liver receptors. In either case Tsou plots suggest that the modification of two arginine residues is responsible for this behavior, although it cannot be ascertained whether the two relevant residues are the same for both receptor types. Circular dichroism studies indicated no apparent changes in protein conformation in the modified hormone. Binding capacity was restored upon regeneration of arginines by incubation with Tris-HCl buffer. Only the carboxy-terminal peptide was isolated by HPLC from a tryptic digest of succinylated Arg-modified hGH, indicating that 183 is the nonreacting arginine residue.
Animals↗
Biochemical characteristics of alveolar macrophage-specific peroxidase activities in the rat.
The biochemical characteristics of endogenous macrophage peroxidases (Po), and their relationship to myeloperoxidase (MPO), have heretofore been poorly understood and were examined in the current study. Rat alveolar macrophages (AM) were homogenized and fractionated by differential centrifugation into lysosomal and microsomal fractions. The Po activities in both fractions were separated using HPLC gel-filtration and two main activities were detected. One, in the lysosomal fraction, had a relative molecular mass (Mr) of 58,000, while the other, associated with the microsomal fraction corresponded to Mr 74,000. By comparison, MPO from rat polymorphonuclear neutrophils (PMN) had Mr 140,000. The 58- and 74-kDa Po activities also differed from MPO with respect to their apparent Km for H2O2 and optimum pH of activity. Using o-dianisidine as a substrate, the Km for H2O2 of the 58- and 74-kDa Po species was 0.4 and 0.19 mM, respectively, compared to 0.011 mM for MPO. Using monochlorodimedon, the corresponding values were 0.22 and 0.195 mM for the 58- and 74-kDa activities and 0.026 mM for MPO. With either substrate, MPO exhibited optimum activity at pH 5.4, compared to 5.2 for the 58-kDa activity and 4.8 for the 74-kDa species. Thus, rat AM contain two endogenous Po activities with biochemical characteristics distinct from those of MPO. Our findings suggest that these activities represent novel peroxidases that may play an important role in the oxidative metabolism of AM.
Animals↗
The action of arginine-specific reagents on ionic and gating currents in frog myelinated nerve.
(1) The effect of arginine-specific reagents on the sodium current (INa), potassium current (IK) and gating current (Igat) of myelinated nerve fibres was investigated. (2) Externally applied camphorquinone-10-sulfonic acid (Cqs-OH) had little effect, but 50 mM Cqs-OH applied to the cut ends of the fibre progressively reduced the amplitude of INa without significantly altering its time course. After 30 min INa was reduced to 52% (pH 9.0) or 66% (pH 6.75-7.6) of the control value. IK was decreased to a similar extent without changing its kinetics. Igat was less affected than the ionic currents. (3) Externally applied phenylglyoxal markedly reduced INa and Igat, but many fibres were lost during or shortly after the treatment. A few min treatment with 5 mM phenylglyoxal at pH 9 reduced INa to 20% and the on-response of Igat to 69.5%. The effect was to a large extent irreversible. (4) External nitrophenylglyoxal and hydroxyphenylglyoxal significantly reduced INa and were less damaging than phenylglyoxal. INa was decreased to 34.5% by 10 mM nitrophenylglyoxal and to 28.3% by 20 mM hydroxyphenylglyoxal. The effect of nitrophenylglyoxal was little reversible, but that of hydroxyphenylglyoxal to a large extent reversible. 20 mM hydroxyphenylglyoxal reduced the on-response of Igat to 62.5% of the control value, i.e. much less than INa. (5) 5 mM phenylglyoxal, 10 mM nitrophenylglyoxal and 20 mM hydroxyphenylglyoxal shifted the steady-state inactivation curve by 10-15 mV to more negative values of membrane potential but did not affect the descending branch of the INa(E) curve. (6) 20-30 mM glyoxal, 20 mM 1,2-cyclohexanedione and 10 mM 4-hydroxy-3-nitrophenylglyoxal had no effect on INa. (7) The results are compatible with the idea that arginine residues are principal components of the sodium channel macromolecule.
Aldehydes↗
An essential arginine residue in human prostatic acid phosphatase.
Treatment of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) with either of the arginine-specific modifiers 2,3-butanedione or 1,2-cyclohexanedione in borate buffer at pH 8.1 leads to loss of activity. The inactivation by cyclohexanedione can be partially reversed by 0.2 M hydroxylamine. The rate of inactivation by both modifiers is decreased in the presence of the competitive inhibitors L-(+)-tartrate or inorganic phosphate but not in the presence of the non-inhibitor D-(-)-tartrate. Amino acid analysis of modified acid phosphatase indicates that only arginines are modified and that L-(+)-tartrate protects at least two arginyl residues from modification. A likely role of these arginyl residues is their involvement in binding the negatively charged phosphate group of the substrate.
Acid Phosphatase↗
Modification of arginyl residues in ferredoxin-NADP+ reductase from spinach leaves.
Reaction of spinach leaves ferredoxin-NADP+ reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) with alpha-dicarbonyl compounds results in a biphasic loss of activity. The rapid phase yields modified enzyme with about 30% of the original activity, but no change in the Km for NADPH. Only partial protection against inactivation is provided by NADP+, NADPH and their analogs, whereas ferredoxin affords complete protection. The reductase inactivated to 30% of original activity shows a loss of about two arginyl residues, whereas only one residue is lost in the NADP+-protected enzymes. The data suggest that the integrity of at least two arginyl residues are requested for maximal activity of ferredoxin-NADP+ reductase: one residue being located near the NADP+-binding site, the other presumably situated in the ferredoxin-binding domain.
Aldehydes↗
Inactivation of adenylate cyclase by phenylglyoxal and other dicarbonyls. Evidence for existence of essential arginyl residues.
Rat brain adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was markedly reduced when the enzyme was preincubated at 23 degrees C for periods up to 30 min with phenylglyoxyal (2.5-20 mM), an agent that binds specifically to arinyl residues. The kinetics indicated the interaction of phenylgloxal with one arginyl residue was responsible for the inactivation. Protection of inactivation of the enzyme by phenylglyoxal was attained in the presence of ATP and to a lesser extent by ADP and 5'-AMP but not by cyclic-AMP or Mg2+. In addition, 2,3-butanedione and 1,2-hexanedione, compounds that also react with arginyl residues, each inactivated adenylate cyclase to varying degrees. Furthermore, this inactivation was enhanced in the presence of borate ions. These observations strongly suggest that brain adenylate cyclase possesses essential arginyl residues.
Adenylyl Cyclase Inhibitors↗
The halide complexes of myeloperoxidase and the mechanism of the halogenation reactions.
The spectral changes caused by the addition of halides to myeloperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) have been investigated and the dissociation constants of the enzyme-halide complexes have been determined. The pH dependence of the dissociation constants suggests that halide binding is associated with a protonation step in myeloperoxidase. Myeloperoxidase catalyzes the peroxidative chlorination and bromination of monochlorodimedone. It is shown that at low pH, chloride acts as a competitive inhibitor with respect to H2O2, whereas at higher pH, H2O2 inhibits the chlorination reaction. The dissociation constant (Kd) of the spectroscopically detectable complex and the Km for chloride are considerably smaller than the inhibition constant (Ki) for chloride. These halogenation reactions are strongly pH dependent, the logarithm of the Km for chloride varies linearly with pH. The position of the pH optimum of the chlorination and bromination reaction is a linear function of the logarithm of the [halide]/[H2O2] ratio. A mechanism of the chlorination and bromination reaction is suggested with substrate inhibition for both hydrogen peroxide and the halide.
Catalysis↗
Chemical modifications of Achromobacter collagenase and their influence on the enzymic activity.
A study of the influence of chemical modifications on the activity of Achromobacter iophagus collagenase (EC 3.4.24.8) has led to the following conclusions: a modification of 4 out of 80 COOH groups with carbodiimide led to 90% loss of enzymic activity. A 70% inactivation was found after modification of two tyrosines out of 30 with tetranitromethane. The modification of four to six tryptophans out of 16 with 2-hydroxy-5-nitrobenzyl bromide decreased enzyme activity to 36%. This inactivation is accelerated in the presence of collagen. An increase of reagent/enzyme molar ratio led to a modification of 16 tryptophan residues and denaturation of Acahromobacter collagenase. A modification of two arginines out of 18 with 1,2-cyclohexanedione and eight NH2 groups out of 24 with 2,3-dimethyl maleic anhydride does not change the collagenolytic activity. All NH2 groups become available for 2,3-dimethyl maleic anhydride after dissociation of the dimer. A possible analogy of hydrolytic site of collagenase with that of two other known bacterial metalloproteinases (thermolysin and Bacillus subtilis neutral proteinase (EC 3.4.24.4)) is discussed.
2-Hydroxy-5-nitrobenzyl Bromide↗