Human milk peroxidase is derived from milk leukocytes

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.     

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.     

Characterization of the hydroperoxide-reducing activity of human plasma

A peroxidase was identified in human plasma using a novel peroxidase assay. In this assay both the substrate 5-phenyl-4-pentenyl hydroperoxide (PPHP) and its reduction product, 5-phenyl-4-pentenyl alcohol (PPA) are quantitated by HPLC. Substrate specificity studies indicated that the peroxidase requires glutathione as reducing substrate. No reduction was detected using the classical heme peroxidase reducing substrates, phenol and hydroquinone. Peroxidase activity was not due to glutathione transferases. Failure to saturate the peroxidase activity with reduced glutathione and inhibition by Cd+2 indicated that it is probably selenium dependent. The enzyme appears to be different from erythrocyte glutathione peroxidase based on kinetic and immunological experiments. The apparent Km values for PPHP are 25 microM for erythrocyte peroxidase and 54 microM for plasma peroxidase at 0.5 mM reduced glutathione. Anti-peroxidase prepared against bovine erythrocyte glutathione peroxidase partially inhibited human erythrocyte peroxidase but did not inhibit human plasma peroxidase.     

Enzymological and immunological studies on a clinical case of platelet cyclooxygenase abnormality

A peroxidase-linked immunoassay of the sandwich type was developed for a quantitative determination of the amount of human cyclooxygenase. Two species of monoclonal antibodies (hPES01 against the human enzyme and PES-5 against the bovine enzyme) were utilized, which recognized different epitopes on the cyclooxygenase of human platelets. The peroxidase activity of the immunoprecipitate was correlated with the amount of cyclooxygenase. The enzyme immunoassay was applied to platelets from 15 normal subjects and a clinical case of platelet cyclooxygenase abnormality with a prolonged bleeding time. Almost the same level of immunoreactive protein was found in platelets of both normal subjects and the patient. However, the solubilized enzyme from the patient's platelets did not transform arachidonic acid to prostaglandin H2 (PGH2) while thromboxane production from PGH2 was observed at a normal level.     

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.     

Identification of immunoreactive sites in bovine parathyroid cells to antibodies raised against the NH2-terminal sequence of parathyroid hormone.

The NH2-terminal sequence of bovine parathyroid hormone (1-84) was localized with different immunocytochemical methods on the light and electron microscopic level in bovine parathyroid glands and in isolated bovine parathyroid parenchymal cells. The peroxidase labeled staphylococcal protein A and the peroxidase anti-peroxidase method were found to be advantageous for light and electron microscopic localization, respectively. Reaction product was found light microscopically in the cytoplasma of the parenchymal cells and electron microscopically largely over the secretion granules of the parenchymal cells. The immunoreactive sites were subsequently identified to represent only intact parathyroid hormone (1-84) by gel electrophoresis derived enzyme linked immunosorbent assay.     

Immunohistochemical localization of phospholipase A2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa.

1. Approximately 150-fold purified phospholipase A2 (PLA2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. Produced antisera blocked PLA2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. Using indirect peroxidase technique PLA2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

Antigenic relation between thyroid peroxidase and the microsomal antigen implicated in auto-immune diseases of the thyroid

Pools of sera from patients with Graves' disease or Hashimoto's thyroiditis highly inhibit the binding to human thyroid membranes of one of 19 monoclonal antibodies raised against preparations of human thyroid membranes. This monoclonal antibody reacts with human and bovine thyroid peroxidase and bovine lactoperoxidase but not with human hemoglobin, cytochrome c and other related molecules. These results indicate that the thyroid peroxidase and the microsomal antigen are antigenically related. These data taken together with those from other groups, highly suggest that thyroid peroxidase is the microsomal antigen involved in autoimmune thyroid diseases.     

Peroxidases in the neural retina and pigment epithelium.

Peroxidase activity, assayed with 2 mM-H₂O₂ and suitable hydrogen donors (either p-phenyl-enediamine or diaminobenzidine), was demonstrated in homogenates of neural retina and pigment epithelium of both the dog and the cow. The enzyme is particle-associated in the native state, but is readily extractable by brief sonication or freeze-thawing. At optimum pH, which is between 4.0 and 4.5 for both sources, the specific activity is up to 40 times greater in pigment epithelial cells than in neural retina. Some catalase activity was detected in extracts from both bovine and canine neural retina, but catalase was essentially absent in pigment epithelium. Fractionation of bovine pigment epithelial cells showed that peroxidase activity is associated mainly with heavy organelles sedimenting at low centrifugal forces. Melanosomes, nuclei, melanolysosomes and plasma membranes were the principal organelles identified in these low speed sediments. It was not possible to separate them either by differential centrifugation or on discontinuous sucrose gradients. However, melanosomes were excluded as the only source of peroxidase activity by isolating separately the melanotic and amelanotic cell populations; equal peroxidase was found in both cell types. Since nuclei are not a likely source of this enzyme, it is suggested that most of the peroxidase activity in bovine pigment epithelial cells is localized in either the melanolysosomes, plasma membranes, or both.     

Preparation of specific antisera to 15alpha-hydroxyestrogens.

The synthesis of haptens of 15alpha-hydroxyestrone, 15alpha-hydroxyestradiol, and 15alpha-hydroxyestriol (estetrol) was undertaken, to obtain specific antisera required for enzyme immunoassay. 3-(1-Carboxypropyl) ethers of these 15alpha-hydroxyestrogens were prepared and conjugated with bovine serum albumin and horseradish peroxidase. The specificity of antisera elicited against bovine serum albumin conjugates was checked by the enzyme immunoassay by using horseradish peroxidase-labeled antigen, and proved to be satisfactory in terms of cross-reactivities to related compounds.     

Purification and characterization of glutathione peroxidase from human blood platelets. Age-related changes in the enzyme

Platelet glutathione peroxidase (GPx) is known to play a pivotal role in controlling the level of lipid hydroperoxides, especially those resulting from the 12-lipoxygenase activity. GPx was purified fromm the cell cytosol by more than 700-fold using an exchange chromatography, FPLP, gel filtration and covalent fixation. Isoelectric focusing revealed a peak activity at pH 5.1. The molecular mass of enzyme was found between 90 and 100 kDa by gel filtration, and was approximating at 23kDa by SDS-PAGE. A polyclonal antibody raised against commercial bovine erthrocyte GPx recognized the human platelet enzyme. It is concluded that human platelet GPx is likely a homotetramer of 92 kDa as described for most sources. We have also found that the decreased platelet GPx activity observed in platelets from elederly people is associated with a lower content of the immunoreactive enzyme.     

Identification of lactoperoxidase in mature human milk

Myeloperoxidase (MPO) derived from milk leukocytes and lactoperoxidase (LPO) secreted from the mammary gland have been identified previously in human colostrum. These peroxidases are known to play host defensive roles through antimicrobial activity. The goals of this study were to measure the peroxidase activity in mature human milk and to characterize the enzyme responsible for the activity. As determined using 3,3',5,5'-tetramethylbenzidine as substrate, whey prepared from human milk samples obtained 1 and 5 months postpartum showed levels of peroxidase activity equivalent to 0.13 +/- 0.18 and 0.24 +/- 0.21 microg/mL bovine LPO (bLPO; n = 13), respectively. Whey from early milk was fractionated into two peaks of peroxidase activity by cation-exchange chromatography; the peroxidase in the first peak was sensitive to dapsone, which is an inhibitor of LPO, whereas the second peroxidase was not. Whey from mature milk showed only the first peak. Purified bLPO and MPO showed chromatographic behaviors that were similar to the first and second peaks, respectively. The dapsone-sensitive peroxidase from mature milk was further purified (952-fold from whey) by hydrophobic interaction chromatography. This preparation showed two bands with molecular masses of 80 and 90 kDa by polyacrylamide gel electrophoresis and immunoblotting using an antibody against bLPO. After deglycosylation, two distinct proteins with lower molecular weights were observed. Amino acid sequencing indicated that both of these proteins are LPO. These results provide evidence that LPO is present in mature human milk and that it is responsible for most of the peroxidase activity in mature milk.     

Binding of estradiol to horseradish peroxidase and horse heart microperoxidase

Horseradish peroxidase and horse heart microperoxidase can bind estradiol to human or bovine serum albumin in the presence of hydrogen peroxide. However, we have shown here that, in the absence of serum albumin, the hormone was fixed by the enzyme molecule itself. Evidence is presented that (a) the hormone is transformed into a water-soluble and dialysable derivative of estradiol; (b) this new product is easily separated from the enzyme by gel filtration chromatography. It appears to have a high affinity for the chromatographic gel. The implications of the binding of an estradiol derivative to peroxidases are discussed.     

Purification and characterization of human salivary peroxidase

Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of inactive or less active forms of tyrosine hydroxylase in human adrenals by a sandwich enzyme immunoassay

A sensitive sandwich enzyme immunoassay for tyrosine hydroxylase (TH) from bovine and human adrenals has been developed. Anti-TH antibody was prepared from bovine adrenal TH. The assay system consisted of an antibody F(ab')2 immobilized on polystyrene beads as a solid phase and of beta-D-galactosidase-conjugated antibody. This method was highly sensitive and specific for the assay of TH. Human adrenal TH level was determined by similar sensitivity as bovine adrenal TH, suggesting the presence of common antigenic sites between human and bovine adrenal enzymes. The presence of inactive or less active forms of TH in human adrenals was revealed by purification of the enzyme and monitoring with this enzyme immunoassay as well as with enzyme activity assay.     

Preparation of neoglycoprotein-enzyme conjugate using a heterobifunctional reagent and its use in solid-phase assays and histochemistry

A conjugate of a neoglycoprotein (chemically lactosylated bovine serum albumin) and an enzyme (horseradish peroxidase) has been prepared in solution using a heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, and has been purified by gel filtration on an Ultrogel AcA-44 column. To preclude any carbohydrate-dependent binding to the sugar residues on the glycoprotein peroxidase, the enzyme has to be treated with sodium periodate and sodium cyanoborohydride prior to coupling, which results in oxidative cleavage of the carbohydrates and reduction of the aldehydes thus formed to primary alcohols. Lactosylated bovine serum albumin-peroxidase conjugate has been employed to detect plastic-bound Ricinus communis agglutinin with dependence of the concentration of the lectin and with dependence of the presence of specific inhibitors. Enzyme-labeled conjugates with unmodified bovine serum albumin are completely ineffective in this assay. Localization of β-galactoside-specific sugar receptors in connective tissue is used to demonstrate the feasibility of application of such neoglycoprotein-enzyme conjugates in histochemistry with a minimum number of steps.     

Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine enzyme.

The glutamate dehydrogenase from a single human liver has been studied. The subunit size was found to be 55,200 +/- 1,500 by sedimentation equilibrium. The partial specific volume is 0.732 as calculated from the amino acid composition. The sequence was determined by isolation of peptides after cyanogen bromide (CNBr) cleavage; the fraction containing the largest peptides was hydrolyzed by trypsin after maleylation. Studies on these peptides accounted for 454 residues of the 505 residues that are presumably present in the protein. For the 51 residues that were not represented in isolated peptides, we have tentatively assumed that the sequence is the same as that of the bovine enzyme. Methionine and arginine residues in these peptides could be placed on the basis of the specificity of cleavage by CNBr or trypsin. In all, 349 residues were placed in sequence, and were aligned by homology with the corresponding peptides of the bovine and chicken enzymes. From the present information, there are 24 known differences in sequence between the human and bovine enzymes and 41 between the human and chicken enzymes. In addition, the human enzyme contains 4 additional residues at the NH2 terminus as compared to the bovine enzyme. In a peptide from the human enzyme, an additional residue, isoleucine 385, was detected by automated Edman degradation. Reinvestigation of the bovine sequence demonstrated that this residue is also present in the bovine enzyme (and presumably in the chicken enzyme also). Residue 384 of the bovine enzyme, previously reported as Glx has now been shown to be glutamine.     

Lens neutral endopeptidase occurs in other bovine and human tissues.

Lens neutral endopeptidase (EC 3.4.24.5) was previously thought to be unique to the eye lens. We report here the finding of a neutral endopeptidase, in a variety of bovine and human tissues, which is very similar both biochemically and immunologically to the lens endopeptidase. SDS/polyacrylamide-gel electrophoresis of partially purified enzyme fractions from various bovine tissues shows the characteristic pattern of at least eight bands with Mr values ranging from 24,000 to 32,000 which was described for the bovine-lens neutral endopeptidase. The relative activity of the enzyme varies from tissue to tissue with lung having the highest activity. Partially purified enzyme fractions from these tissues cross-react with antiserum raised in rabbit against bovine lens endopeptidase showing apparent identity when examined side by side in Ouchterlony double-diffusion tests. The human enzyme also cross-reacts with the antiserum but when tested by double-diffusion against the bovine enzyme the precipitin lines show spurring at the joining edges indicating a structural difference between the human and the bovine enzymes. It was also found by Western blot experiments, after denaturing polyacrylamide-gel electrophoresis of the enzyme, that the polypeptide components of the human and bovine enzymes show somewhat different banding patterns.