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At least 19 recordsLinked to original sources
PubMed2001

Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase.

BACKGROUND: Two heme-containing peroxidases, secretory lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased. OBJECTIVE: Our knowledge of lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of lactoperoxidase. DESIGN: Polyclonal antibodies were raised against recombinant human lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively. RESULTS: Two bands with molecular masses of approximately 80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate-polyacrylamide gel electophoresis profile of purified lactoperoxidase. The mean (+/-SD) concentration of lactoperoxidase in 26 whey samples was estimated to be 0.77 +/- 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples. CONCLUSION: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.

Antibodies↗
PubMed1979

Lactoperoxidase activity in guinea-pig milk and saliva: correlation in milk of lactoperoxidase with bactericidal activity against Escherichia coli.

The lactoperoxidase (LPO) activity in guinea-pig milk and saliva has been investigated in sows suckling normal young, and young orally infected with Escherichia coli. There was a 5-fold increase in activity in milk during the 3--4 weeks of lactation; infection of the young did not alter this. There was no comparable increase in lactoperoxidase activity of saliva during this same period, either in the infected or non-infected group. The antibacterial activity of milk from sows suckling normal young increased with the lactoperoxidase, and this bactericidal activity could be reversed by LPO inhibitors such as penicillamine and cysteine but not by addition of sufficient iron to saturate the lactoferrin. In milk from sows suckling infected young, bacteriostatic activity occurring in samples from about 14 days after infection needed iron or both iron and penicillamine (or cysteine) for reversal, indicating that both the antibody-lactoferrin system and the LPO system may be involved in the infected state.

Animals↗
PubMed1987

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase.

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.

Animals↗
PubMed2004

Protein radical formation during lactoperoxidase-mediated oxidation of the suicide substrate glutathione: immunochemical detection of a lactoperoxidase radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct.

A novel anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes protein radical-derived DMPO nitrone adducts has been developed. In this study, we employed this new approach, which combines the specificity of spin trapping and the sensitivity of antigen-antibody interactions, to investigate protein radical formation from lactoperoxidase (LPO). When LPO reacted with GSH in the presence of DMPO, we detected an LPO radical-derived DMPO nitrone adduct using enzyme-linked immunosorbent assay and Western blotting. The formation of this nitrone adduct depended on the concentrations of GSH, LPO, and DMPO as well as pH values, and GSH could not be replaced by H(2)O(2). The level of this nitrone adduct was decreased significantly by azide, catalase, ascorbate, iodide, thiocyanate, phenol, or nitrite. However, its formation was unaffected by chemical modification of free cysteine, tyrosine, and tryptophan residues on LPO. ESR spectra showed that a glutathiyl radical was formed from the LPO/GSH/DMPO system, but no protein radical adduct could be detected by ESR. Its formation was decreased by azide, catalase, ascorbate, iodide, or thiocyanate, whereas phenol or nitrite increased it. GSH caused marked changes in the spectrum of compound II of LPO, indicating that GSH binds to the heme of compound II, whereas phenol or nitrite prevented these changes and reduced compound II back to the native enzyme. GSH also dose-dependently inhibited the peroxidase activity of LPO as determined by measuring 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) oxidation. Taken together, these results demonstrate that the GSH-dependent LPO radical formation is mediated by the glutathiyl radical, possibly via the reaction of the glutathiyl radical with the heme of compound II to form a heme-centered radical trapped by DMPO.

Amino Acids↗
PubMed1983

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.

Animals↗
PubMed1980

Lactoperoxidase-tubulin interactions.

Self-iodinated lactoperoxidase co-polymerizes with brain microtubules to constant specific activity and a stoichiometry of 0.2 to 0.3 lactoperoxidase molecule/tubulin heterodimer polymerized in the presence of 4 M glycerol. By contrast, iodinated tubulin loses its competence to polymerize. The lactoperoxidase-microtubule association is salt- and temperature-sensitive, shows considerable specificity, is saturable, and is reversible. Lactoperoxidase does not displace the microtubule-associated proteins from microtubules, does not promote polymerization, and binds to preformed microtubules. Self-iodinated lactoperoxidase also binds to tubulin oligomers at 0 degrees C and in the presence of CaCl2. The stoichiometry for this interaction is 0.6 to 0.8 molecules of enzyme/dimer. Lactoperoxidase forms a complex with soluble brain tubulin prepared by two cycles of polymerization and depolymerization or by phosphocellulose chromatography. The interaction was studied by sucrose gradient analysis, gel filtration, and spectral analysis based on the finding that tubulin binding to lactoperoxidase leads to a red shift in the Soret spectrum, yielding a difference spectrum with a minimum of 410 nm and maximum at 430 nm. This interaction involves one or more sulfhydryl groups of tubulin. Complex formation is relatively slow, is retarded by 0.6 M NaCl, and is accelerated by diiodotyrosine. By all three methods of analysis, the stoichiometry approaches a value of 2 lactoperoxidase molecules/tubulin dimer. There is a single class of binding sites in pig, beef, or rat tubulin with an apparent overall affinity constant of approximately 2.0 x 10(6) M-1. The molecular weight of the complex by sucrose gradient or gel filtration is approximately 140,000 i.e. half of the expected value for a 2:1 adduct. Since both alpha and beta subunits are present in the complex, we propose that the complex consists of a mixture of equal parts of presumably native alpha-tubulin-lactoperoxidase and beta-tubulin-lactoperoxidase.

Animals↗
PubMed2005

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.

Bacterial Proteins↗
PubMed2002

Variations of lactoperoxidase activity and thiocyanate content in cows' and goats' milk throughout lactation.

Lactoperoxidase activity and thiocyanate content were monitored in the milks of seven individual cows and six goats during lactation. Lactoperoxidase activity exhibited a cyclic pattern with alternating peaks and troughs throughout lactation. Extremely large variations were observed between and within animals. For example, lactoperoxidase activity ranged from 0.05-5.60 U/ml for one cow on different sampling days. Variations between cows were also large, ranging from 0.98-5.1 U/ml on a specific sampling day. Mean lactoperoxidase activity in cows ranged from 1.5-2.7 U/ml with an overall mean of 2.3 +/- 1.0 U/ml while the thiocyanate concentration ranged from 6.0-10.2 micrograms/ml with an overall average of 8.5 +/- 5.1 micrograms/ml. Lactoperoxidase activity means for goats ranged from 0.04-0.16 U/ml with a grand mean of 0.1 +/- 0.06 U/ml while the thiocyanate content means ranged from 6.6-8.2 micrograms/ml with a grand mean of 7.0 +/- 2.59 micrograms/ml. There was no correlation between lactoperoxidase activity and thiocyanate content in either cows' (R2 = 0.011) or goats' (R2 = 0.015) milk. These experiments have revealed that lactoperoxidase activity is affected by many factors including the individual animals, species, feed and stage of lactation. Therefore the exogenous supply of thiocyanate and hydrogen peroxide needed to activate the lactoperoxidase system for raw milk preservation will vary in quantity depending on these factors.

Animals↗
PubMed2000

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.

Animals↗
PubMed2001

Analysis time and lactation stage influence on lactoperoxidase system components in dairy ewe milk.

To study the effect of time elapsed from the moment of taking samples on lactoperoxidase system components, we analyzed the activity of the lactoperoxidase enzyme and the concentrations of thiocyanate and hydrogen peroxide in 46 individual samples of Manchega ewe milk. Samples were maintained at a temperature of 4 degrees C until analysis, which took place at 6, 12, 24, and 48 h after extraction. Decreases were observed in lactoperoxidase activity when the analyses were performed at 48 h and in the thiocyanate and hydrogen peroxide concentrations at 12 h compared with those carried out earlier. Consequently, when the components of the lactoperoxidase system or its antibacterial activity are studied, the time elapsed since the sampling commenced must be taken into account. Similarly, the time elapsed is important when carrying out bacterial counts or residue screening by microbiological methods, during which the lactoperoxidase system may interfere. To study the component changes in the lactoperoxidase system during lactation, samples obtained 15, 30, 45, 60, 75, 90, 105, 120, and 135 d postpartum from 48 Manchega ewes were used. Average lactoperoxidase activity, thiocyanate, and hydrogen peroxide concentrations were 3.46 U/ml, 6.89 mg/L, and 0.39 mg/L, respectively, with significant variations throughout lactation. The thiocyanate and hydrogen peroxide levels at different lactation stages seemed to be insufficient to activate the lactoperoxidase system. Nevertheless, this could be achieved by adding 5 mg/L of thiocyanate and 8 mg/L of hydrogen peroxide at any time during lactation.

Animals↗
PubMed2000

Bovine lactoperoxidase and its recombinant: comparison of structure and some biochemical properties.

Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.

Animals↗
PubMed1984

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.

Animals↗