The inactivation of lactoperoxidase and the acyl phosphatase activity of oxidized glyceraldehyde-3-phosphate dehydrogenase by phenylhydrazine and phenyldiimide

The acyl phosphatase activity catalyzed by the sulfenic acid form of glyceraldehyde-3-phosphate dehydrogenase (GPD) is inactivated by phenylhydrazine, isopropylhydrazine, and phenyldiimide under anaerobic conditions. The hydrazines reactivate the dehydrogenase function of GPD and, therefore, reduce the sulfenic acid at the active site of the acyl phosphatase. Lactoperoxidase is also inactivated by phenylhydrazine, isopropylhydrazine, and phenyldiimide under anaerobic conditions. When lactoperoxidase is inactivated by an aerobic, aqueous solution of [¹⁴C] phenylhydrazine 1 mole of phenylhydrazine is covalently bound per 40,000 g of lactoperoxidase.     

Structure-potential dependence of adsorbed enzymes and amino acids revealed by the surface enhanced Raman effect

Surface enhanced Raman scattering (SERS) of some enzymes (alkaline phosphatase, horseradish peroxidase and lactoperoxidase) and some amino acids (tryptophan, tyrosine and phenylalanine) on silver electrodes has been studied. The spectral band intensities of certain amino acids and amino acid residues were determined by their orientation on the surface and depended on the electrode potential (E).Abbreviations SERS surface enhanced Raman scattering - Trp tryptophan - Tyr tyrosine - Phe phenylalanine - E electrode potential - ORC oxidation-reduction cycle     

Identification, purification, and characterization of a non-heme lactoperoxidase in bovine milk

A purification procedure for a protein related to lactoperoxidase devoid of the heme prosthetic group under conditions also yielding enzymatically active lactoperoxidase is described. These two forms were separated from bovine milk according to their respective behaviors on cation exchange. Lactoperoxidase and non-heme lactoperoxidase had the same apparent molecular weight in the denatured (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and native form (velocity sedimentation on sucrose gradient) about 85,000; but unlike lactoperoxidase, non-heme lactoperoxidase was devoid of light absorption properties in the Soret region and of enzyme activity. Lactoperoxidase and non-heme lactoperoxidase contained a similar amount of carbohydrate and gave very similar peptide maps after limited proteolysis by subtilisin or trypsin. The two forms appeared to be immunologically related since they gave a single line in immunodiffusion using anti-lactoperoxidase antibodies and since 125I-labeled non-heme lactoperoxidase and 125I-labeled lactoperoxidase reacted with anti-lactoperoxidase antibodies in radioimmunoassay. Lactoperoxidase and nonheme lactoperoxidase were compared in their ability to interact with diiodotyrosine and tubulin (Rousset, B., and Wolff, J. (1980) J. Biol. Chem. 255, 2514-2523). 125I-labeled diiodotyrosine bound specifically to lactoperoxidase. No detectable binding has been observed with nonheme lactoperoxidase. In contrast, lactoperoxidase and non-heme lactoperoxidase coupled to an insoluble matrix were able to bind rat brain tubulin, indicating that both forms of lactoperoxidase can be used for an affinity chromatography purification procedure of brain tubulin. Non-heme lactoperoxidase was found in milk from several origins, cow, goat, sheep, and human. In bovine milk, lactoperoxidase and non-heme lactoperoxidase were found in comparable amounts.     

Visualization of lactoperoxidase binding to microtubule and tubulin

The binding of lactoperoxidase to microtubules and tubulin was shown in both electron micrography and polyacrylamide gel electrophoresis by tracing the enzymatic activity of lactoperoxidase. Lactoperoxidase bound to purified microtubules appeared to distribute evenly on the surface without forming special structures. Both alpha and beta-tubulin separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis bound lactoperoxidase, and could be detected by the use of lactoperoxidase reaction. Electrophoretic study revealed that the interaction between lactoperoxidase and tubulin were not strictly specific and a variety of proteins other than alpha- and beta-tubulin, including actin and neurofilament subunits, bound lactoperoxidase.     

Interaction of lactoperoxidase with thiols and diiodotyrosine.

Glutathione and cysteine bind to the heme of lactoperoxidase, thereby causing a red shift of the Soret band which is reversed upon addition of iodide or guaiacol, two substrates for lactoperoxidase. The rate of formation of the enzyme-thiol complex is enhanced by diiodotyrosine. Binding of diiodotyrosine to lactoperoxidase does not cause a shift of the Soret band which indicates binding to the protein of the enzyme. At neutral pH and low ionic strength, lactoperoxidase is adsorbed on insolubilized diiodotyrosine (diiodotyrosine-agarose). It can be eluted at slightly increased ionic strength which shows that the binding is weak. In the presence of 5 X 10(-4) M glutathione, however, the binding of the enzyme to diiodotyrosine-agarose becomes much stronger so that a high salt concentration is required for elution. Lactoperoxidase is also adsorbed on insolubilized thiols (thiol-agarose). The presence of diiodotyrosine is not required for strong binding. A simple method for the preparation of lactoperoxidase from milk by affinity chromatography is based on the interactions of the enzyme with the two ligands, thiols and diiodotyrosine.     

Iodide binding and regulation of lactoperoxidase activity toward thyroid goitrogens.

The effects of the antithyroid goitrogens, methylthiouracil and methylmercaptoimidazole, on the oxidation of N-acetyltyrosylamide at pH 8.8 by lactoperoxidase have been evaluated in the presence and the absence of iodide for the purpose of elucidating the effects of iodide. At pH 8.8, iodine is not oxidized. In the absence of iodide, the two antithyroid drugs inactivate lactoperoxidase by a second order process. When iodide is added before methylthiouracil or methylmercaptoimidazole, enzyme inactivation does not occur as rapidly and both goitrogens are readily oxidized. The kinetics of the oxidation reactions have been analyzed in order to obtain the equilibrium constant of the iodide . lactoperoxidase complex. Essentially the same iodide dissociation constant, i.e. 2 x 10(-5) M, was found by studying its effects on the kinetics of oxidation of the two antithyroid drugs. A large difference absorption spectrum is observed in the Soret region between native lactoperoxidase and lactoperoxidase inactivated by methylthiouracil.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Enzymatic Iodination of Sindbis Virus Proteins

Sindbis virus was iodinated by using the enzyme lactoperoxidase, an iodination technique which labels only surface proteins. By this technique, the two viral glycoproteins are labeled, and the internal viral protein is not. The two glycoproteins are iodinated to strikingly different extents. This difference in susceptibility to iodination apparently is due to the position or conformation of the glycoproteins in the envelope spikes of the virion and not to differing contents of tyrosine, the amino acid substrate of lactoperoxidase. Both viral glycoproteins are iodinated by lactoperoxidase on the surface of Sindbis-infected chicken cells. Here, as in the virion, the glycoproteins are iodinated unequally, with the smaller glycoprotein again being preferentially iodinated. Another virus-specific protein found in large amounts in infected cells, and from which the preferentially iodinated virion glycoprotein is produced by a proteolytic cleavage, is not iodinated by lactoperoxidase. Thus it appears that the viral glycoproteins are present on the cell surface and that the precursor protein is not.     

Interaction of lactoperoxidase with enzymes and immunoglobulins in bovine milk

The interaction of lactoperoxidase with lysozyme and ribonuclease as well as immunoglobulins from cow milk has been investigated. As gel filtration and enzyme kinetics experiments have shown, the lactoperoxidase was slightly activated by complexing to lysozyme, while IgA and IgM were inhibitory for the peroxidase. Oh the other hand, IgG and ribonuclease had no effect on the enzyme activity although the latter did form a complex with the lactoperoxidase. The interaction between the lysozyme and lactoperoxidase appears to be rather specific since the alteration of the lactoperoxidase sugar moiety by periodate oxidation, prevented the formation of the lactoperoxidase-lysozyme complex.     

Downstream processing of lactoperoxidase from milk whey by involving liquid emulsion membrane

The current work deals with downstream processing of lactoperoxidase using liquid emulsion membrane from the bovine milk whey, which is a by-product from dairy industry. It is an alternate separation technique that can be used for the selective extraction of lactoperoxidase. The extraction of lactoperoxidase in liquid emulsion membrane takes place due to the electrostatic interaction between the enzyme and polar head group of reverse micellar surfactant. The optimum conditions resulted in 2.86 factor purity and activity recovery of 75.21%. Downstream processing involving liquid emulsion membrane is a potential technique for the extraction of lactoperoxidase from bovine whey.     

Optical spectra of lactoperoxidase as a function of solvent

The iron of lactoperoxidase is predominantly high-spin at ambient temperature. Optical spectra of lactoperoxidase indicate that the iron changes from high-spin to low-spin in the temperature range from room temperature to 20 K. The transformation is independent of whether the enzyme is in glycerol/water or solid sugar glass. Addition of the inhibitor benzohydroxamic acid increases the amount of the low-spin form, and again the transformation is independent of whether the protein is in an aqueous solution or a nearly anhydrous sugar. In contrast to lactoperoxidase, horseradish peroxidase remains high-spin over the temperature excursion in both solvents and with addition of benzohydroxamic acid. We conclude that details of the heme pocket of lactoperoxidase allow ligation changes with temperature that are dependent upon the apoprotein but independent of solvent fluctuations. At low pH, lactoperoxidase shows a solvent-dependent transition; the high-spin form is predominant in anhydrous sugar glass, but in the presence of water, the low-spin form is also present in abundance. The active site of lactoperoxidase is not as tightly constrained at low pH as at neutrality, though the enzyme is active over a wide pH range.     

Oxidation of the substituted catechols dihydroxyphenylalanine methyl ester and trihydroxyphenylalanine by lactoperoxidase and its compounds

The reactions of native lactoperoxidase and its compound II with two substituted catechols have been investigated by ESR spin stabilization and spin trapping and by rapid scan and conventional spectrophotometric techniques. The catechols are Dopa methyl ester (dihydroxyphenylalanine methyl ester) and 6-hydroxy-Dopa (trihydroxyphenylalanine). o-Semiquinone radicals are formed in the anaerobic reaction of Dopa methyl ester with hydrogen peroxide catalyzed by native lactoperoxidase. The comparable anaerobic reaction of 6-hydroxy-Dopa appears to produce hydroxyl radicals in an unusual reaction. Compound II is reduced back to native lactoperoxidase by both catechols. The reaction between Dopa methyl ester and compound II undergoes an oscillation. The results on the overall lactoperoxidase cycle indicate two successive one-electron reductions of the peroxidase intermediates back to the native enzyme. The resulting free radical formation of o- and p-semiquinones and subsequent formation of stable quinones and Dopachromes is dependent upon the stereochemical arrangement of the catechol hydroxyl groups.     

Inactivation of Escherichia coli and Listeria innocua in milk by combined treatment with high hydrostatic pressure and the lactoperoxidase system

We have studied inactivation of four strains each of Escherichia coli and Listeria innocua in milk by the combined use of high hydrostatic pressure and the lactoperoxidase-thiocyanate-hydrogen peroxide system as a potential mild food preservation method. The lactoperoxidase system alone exerted a bacteriostatic effect on both species for at least 24 h at room temperature, but none of the strains was inactivated. Upon high-pressure treatment in the presence of the lactoperoxidase system, different results were obtained for E. coli and L. innocua. For none of the E. coli strains did the lactoperoxidase system increase the inactivation compared to a treatment with high pressure alone. However, a strong synergistic interaction of both treatments was observed for L. innocua. Inactivation exceeding 7 decades was achieved for all strains with a mild treatment (400 MPa, 15 min, 20 degrees C), which in the absence of the lactoperoxidase system caused only 2 to 5 decades of inactivation depending on the strain. Milk as a substrate was found to have a considerable effect protecting E. coli and L. innocua against pressure inactivation and reducing the effectiveness of the lactoperoxidase system under pressure on L. innocua. Time course experiments showed that L. innocua counts continued to decrease in the first hours after pressure treatment in the presence of the lactoperoxidase system. E. coli counts remained constant for at least 24 h, except after treatment at the highest pressure level (600 MPa, 15 min, 20 degrees C), in which case, in the presence of the lactoperoxidase system, a transient decrease was observed, indicating sublethal injury rather than true inactivation.     

On the ability of lactoperoxidase to catalyze the peroxidase-oxidase oxidation of a vitamin E water-soluble derivative (Trolox C).

The rapid-scan spectral technique has been applied to test conversion of the lactoperoxidase compounds during the peroxidase-oxidase catalyzed oxidation of Trolox. The results clearly indicate a normal peroxidatic pathway of Trolox degradation. Changes of spectral scan profiles were investigated to study directly the interaction of Trolox with lactoperoxidase compound III. Oxygen radicals were not involved in peroxidase-mediated oxidation of Trolox. The rate of the one-electron reduction of lactoperoxidase compound I to II was the same in the absence and presence of equal amounts of Trolox and ascorbic acid, which is also a good substrate for lactoperoxidase. The oxidation of Trolox by lactoperoxidase has potential physiological relevance. Since it could help maintain the catalytic cycles and activity of animal peroxidases, leading to detoxification of hydrogen peroxide as a main product of inflammation processes.     

Reduction of lactoperoxidase by the dithionite anion monomer

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped-flow spectrophotometry in an anaerobic system. Under pseudo-first-order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO2- as the reducing species. The second-order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase. The second-order rate constant for the reduction of lactoperoxidase at pH 7.0 (kappa 1 = 1.3 X 10(5) M-1 s-1) was about three times higher than the rate constant for the reduction of cyanide-bound lactoperoxidase and two times the rate constant for the reduction of the fluoride-lactoperoxidase complex.     

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.     

Action of the lactoperoxidase system on Salmonellae and Shigellae

In vitro experiments have demonstrated that the lactoperoxidase system produces a bactericidal effect on salmonellae and shigellae. The physiological concentrations of the components of this system, making it possible to obtain a pronounced bactericidal effect, have been established. Lactobacilli have been shown to potentiate the effect of the lactoperoxidase system. The possibility of realizing the bactericidal properties of the lactoperoxidase system with respect to salmonellae and shigellae in the preparations of immune lactosera, intended for passive enteral immunization against intestinal infections, has been suggested.     

Assignment of the axial ligands of the haem in milk lactoperoxidase using magnetic circular dichroism spectroscopy

The absorption and MCD spectra of ferric lactoperoxidase from milk and its cyanide and fluoride derivatives have been measured in the near infrared and visible wavelength regions both at room temperature and at 4.2 K. By comparison with the MCD spectra of haemoproteins of known axial ligation, which also contain protohaem IX, it has been possible to arrive at suggestions for the axial ligation in lactoperoxidase. At room temperature oxidized lactoperoxidase has the haem iron in the high-spin state, and the results indicate that the proximal ligand of the haem iron is a histidine imidazole and that the sixth ligand is probably a carboxylate ion. At 4.2 K oxidized lactoperoxidase converts almost totally to a low-spin form, changing the sixth ligand to a histidine imidazole, which is in the imidazolate form.     

Primary structure of bovine lactoperoxidase, a fourth member of a mammalian heme peroxidase family

Much is known about bovine lactoperoxidase but no data are available on its primary structure. In this work its main active fraction was isolated from cow's milk and sequenced using a conventional strategy. A clear similarity was found with human myeloperoxidase, eosinophil peroxidase and thyroperoxidase, the sequences of which were recently elucidated from those of their cDNAs and/or genes. The single peptide chain of bovine lactoperoxidase contains 612 amino acid residues, including 15 half-cystines and 4 or 5 potential N-glycosylation sites. The corresponding peptide segments of human myeloperoxidase, eosinophil peroxidase and thyroperoxidase display 55%, 54% and 45% identity with bovine lactoperoxidase, respectively, with 14 out of the 15 half-cystines present in each of the four enzymes being located in identical positions. The occurrence of an odd number of half-cystines in bovine lactoperoxidase supports the recent finding of a heme thiol released from this enzyme by a reducing agent, suggesting that the heme is bound to the peptide chain via a disulfide linkage, since the absence of free thiol in the enzyme was reported long ago.