Tissue distribution of constitutive and induced soluble peroxidase in rat. Purification and characterization from lacrimal gland.

A thorough search for a soluble peroxidase in 31 different tissues of rat indicated the presence of a constitutive activity only in lacrimal, preputial and submaxillary gland. An induced soluble peroxidase activity was also detected in the lactating mammary gland and in the estrogen-induced uterine secretory fluid. The lacrimal gland was the richest source of the enzyme. No peroxidase activity was detected in the lactating mammary gland of mouse and hamster nor in the preputial gland of mouse and uterine fluid of hamster. The three constitutive and two induced soluble peroxidases of rat had a native molecular mass of 73 kDa by gel filtration and they showed a similar mobility in native PAGE. Lactoperoxidase of cow's milk and solubilized rat membrane-bound peroxidases of uterus, intestine and bone marrow showed in native PAGE a mobility which was distinctly different from that of rat soluble peroxidases. As the lacrimal gland of rat was the richest source of soluble peroxidase, the enzyme was purified from this gland to apparent homogeneity; SDS/PAGE then showed a single band of molecular mass 75 kDa which was similar to that obtained by gel filtration. Peroxidase also purified from preputial and submaxillary gland, as well as commercial lactoperoxidase, had a similar molecular mass on SDS/PAGE to purified lacrimal peroxidase. The visible spectrum of lacrimal peroxidase was similar to that of lactoperoxidase but different from membrane-bound peroxidase of rat neutrophils. On isoelectric focussing, purified lacrimal peroxidase resolved into about 14 multiple forms spanning a pI range of 6.5-3.5 while lactoperoxidase focussed at the cathode. Evidence presented suggests that the multiple forms are possibly due to differences in glycosylation. Immunodiffusion, immunoprecipitation and Western blot using antilacrimal peroxidase serum showed a similar interacting species for all five soluble peroxidases of rat while membrane-bound peroxidases showed no interaction. Although in immunodiffusion, the antiserum failed to cross-react with lactoperoxidase it did interact with lactoperoxidase on Western blot. The results indicate that the various constitutive and induced soluble peroxidases of rat tissues are similar to lacrimal peroxidase but are distinctly different from the known membrane-bound peroxidases of rat. However the lacrimal peroxidase shows both similarities as well as dissimilarities with bovine lactoperoxidase. This soluble peroxidase system of rat could be useful to study tissue-specific regulation of gene expression at the molecular level.     

Purification and characterization of a soluble peroxidase of rat preputial gland: comparison with lactoperoxidase.

A highly active soluble peroxidase (donor: H2O2 oxidoreductase EC 1.11.1.7) has been purified from the preputial gland of the rat by hydroxylapatite chromatography, ammonium sulfate fractionation, Sephadex gel filtration and affinity chromatography on con A-Sepharose. The enzyme shows apparent homogeneity when analysed by acid and alkaline-PAGE. Its molecular, spectral, kinetic and catalytic properties were compared with those of bovine lactoperoxidase (LPO). Preputial gland peroxidase (PPO) is a glycoprotein of molecular weight of 70-73 kDa slightly lower (78 kDa) than that of LPO. Using isoelectric focussing, PPO was resolved into eight different closely spaced protein species spanning a pI range of 5.4 to 6.4, while LPO focuses into several closely spaced protein bands in the pI range 8.5-9.3. PPO is similar to LPO in its spectral (Soret) and some kinetic properties, but it differs significantly from LPO in substrate (H2O2) tolerance and substrate inactivation. PPO also differs from LPO in showing differential inactivation by SDS. Both enzymes are glycoproteins and although concanavalin A (con A) showed a variable interaction with both enzymes, wheat germ agglutinin interacted specifically with LPO only. We suggest that PPO, the secretory peroxidase of the preputial gland, differs significantly from LPO in its molecular and catalytic properties.     

Uterine changes on removal of submaxillary glands in rats (histological changes/uterine peroxidase/blood estradiol level)

Surgical removal of submaxillary gland in immature rats causes a large increase in size and about three to four fold increase in dry and wet weight of uterus compared to that of the sham operated animals of the same age group. Histological examination reveals a significant increase in the diameter of the uterus with considerable elongation of the luminal epithelium from cubical to columnar in the experimental group. Biochemical studies show that the uterine peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), a marker enzyme for uterine growth, increases by ten to fifteen fold on submaxillariectomy and returns almost to the normal level on administration of submaxillary gland extract (105,000 X g supernatant) to the submaxillariectomized animals. Estrogen estimation by radioimmunoassay shows a similar increase of three to four fold on removal of submaxillary glands and decrease almost to the normal value on administration of the submaxillary extract.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.     

Salivary peroxidases

Summary Peroxidases are known to be involved in the intracellular metabolism of H₂O₂ coupled with various physiological functions. Apart from the thyroid gland, the enzyme has been isolated from various extrathyroidal sources of which salivary gland is one of the richest sources of the enzyme. The enzyme from bovine and goat submaxillary gland has been extensively studied in terms of their molecular, spectral, kinetic, catalytic and immunological properties and compared with the lactoperoxidase which is similar to the salivary peroxidase. The modulation of the salivary peroxidase by various factors and the probable mechanism of the modulation has been described. The enzyme has also been compared with the thyroid peroxidase as regards their physicochemical properties as well as on the immunological and functional aspects. The similarities and dissimilarities have been incorporated. The possible function of the enzyme in iodine metabolism and in bactericidal action has been discussed.[/p]     

Purification and Partial Characterization of Tomato Extensin Peroxidase

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.     

Identification of epidermal growth factor in mouse abdominal effusion

Epidermal growth factor (mEGF)-like immunoreactive material(s) was identified in mouse abdominal effusion (approximately 2.1 ng/mg protein) by our enzyme immunoassay (EIA) for mEGF. This material(s) and mEGF from the submaxillary glands of male mice were virtually equivalent with respect to the molecular weight and the antigenicity. Also, on isoelectric focusing analysis, the mEGF-like material(s) identified in abdominal effusion gave a major peak at pH 4.2 and a minor one at pH 4.5. These results demonstrate that the mEGF-like material(s) found in abdominal effusion is a molecule identical to mouse submaxillary gland EGF. Further we found that sialoadenectomy did not cause a marked decrease in the level of mEGF in abdominal effusion, suggesting that the source of mEGF found in abdominal effusion is other than the submaxillary glands.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Peroxidase-mediated transformation of hydroxy-9,10-anthraquinones

A peroxidase (EC 1.11.1.7) has been isolated and purified from Senna angustifolia. The enzyme was purified by ion-exchange chromatography on high Q and high S columns. SDS-PAGE electrophoresis showed that the protein has a molecular mass of approximately 70 kDa. Hydroxy-anthraquinones and hydroxy-anthracenones were evaluated as substrate of S. angustifolia and horseradish peroxidases. Both peroxidases catalyzed the oxidation of alizarin and purpurin anthraquinones to the corresponding 3,3'-bializarin and the new compound 3,3'-bipurpurin, respectively, as well as the formation of 2,2'-biquinizarin from quinizarin anthracenone. The K(Mapp) and V(max) values for alizarin and purpurin were 97 and 95 microM, and 1.5 and 2.1 microM min(-1) mg prot(-1), respectively. The results suggest that peroxidase may participate in the biogenesis of anthraquinones.     

Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.     

Purification and characterization of arginase from Neurospora crassa

We have purified an enzymatically active form of arginase from a wild-type strain of Neurospora crassa to homogeneity. The enzyme has a subunit molecular weight of 38,300 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native protein migrated as a hexamer during gel-filtration chromatography with an apparent molecular weight of 266,000. The enzyme exhibited hyperbolic kinetics at pH 9.5 with an apparent Km for arginine of 131 mM. Antiserum was prepared against the purified enzyme and used to demonstrate the existence of three cross-reactive proteins in crude extracts of wild-type N. crassa. One of these proteins corresponded to the purified protein, whereas the other two were of molecular weights 41,700 and 26,800, respectively. Using the same antiserum, we found that rat liver, but not rat kidney, contains immunoreactive material. We also detected two proteins in extracts of Saccharomyces cerevisiae that were weakly cross-reactive with the antiserum. These data provide evidence for the existence of multiple forms of arginase in fungi as well as in mammals.     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

Studies on submaxillary gland immunoreactive glucagon.

Biologically active immunoreactive glucagon is present in submaxillary gland of rat, mouse, guinea pig and human and can be extracted by saline adjusted to pH 2.8 with HCl. Chromatography on Sephadex G-150 indicates its molecular weight to be 29,000. It has similar immunologic characteristics as pancreatic glucagon. It is biologically active and elevates plasma glucose and insulin when injected intraperitoneally into rats. Compared to pancreatic glucagon, the hyperglycemic effect persists much longer. It competes with pancreatic glucagon for binding to specific glucagon receptors of rat liver plasma membranes. It is stable to pH changes, however, urea dissociates it into several smaller molecular weight fragments including that of 3500. It appears to be an aggregate of smaller glucagon molecules and is not responsible for immunoreactive glucagon in totally eviscerated rats. $\text{In vitro}$, the submaxillary gland does not release immunoreactive glucagon in response to arginine or glucose.     

Tissue kallikrein synthesis and its modification by testosterone or low dietary sodium.

A method has been developed to measure the relative rate of rat tissue kallikrein synthesis which employs a specific antiserum raised against a purified rat urinary kallikrein. Incorporation of [35S]methionine into kallikrein and protein 20 min after intraperitoneal injection was measured in submaxillary gland, pancreas, kidney and descending colon. Kallikrein content was measured with a direct radioimmunoassay, and kallikrein-specific incorporation of [35S]methionine measured after immunoprecipitation. Kallikrein specific radioactivity (c.p.m./mg of enzyme) was about 100-fold greater than that in total protein in both kidney and colon. In contrast, in pancreas the incorporation into the enzyme was only 5-fold higher than into protein, and in submaxillary gland the incorporation was equivalent. Measured as kallikrein-specific radioactivity relative to total protein radioactivity incorporated in 20 min, kallikrein represents 0.18% of total protein synthesis in the kidney, 0.34% in the pancreas, 0.41% in the colon, but 7.29% in the submaxillary gland. Dietary Na+ restriction increased the relative rate of kallikrein synthesis 1.8-fold in the kidney without a comparable effect in submaxillary gland. In contrast, testosterone increased the relative rate of synthesis 2.3-fold in submaxillary gland, but decreased it in kidney. The data show that endogenous kallikrein synthesis differs markedly in various tissues, and that interventions which are known to change kallikrein content or excretion also change the relative rate of enzyme synthesis.     

Identification of the mucin core protein by cell-free translation of messenger RNA from bovine submaxillary glands

Bovine submaxillary mucin was purified and subjected to chemical deglycosylation by treatment at 20 degrees C with either anhydrous hydrogen fluoride or trifluoromethane sulfonic acid. Virtually all of the sialic acid, galactose, fucose, and over 90% of the N-acetylhexosamines were removed by these treatments. The amino acid compositions of the deglycosylated and native mucins were similar indicating that chemical deglycosylation did not cause significant degradation of the protein. Antiserum specific for the deglycosylated bovine submaxillary mucin was produced by immunization of rabbits with the deglycosylated mucin. RNA was isolated from bovine submaxillary glands by extraction with guanidine hydrochloride and further fractionated by chromatography on oligo(dT)-cellulose to yield poly(A)+ mRNA. The poly(A)+ mRNA was translated in a rabbit reticulocyte cell-free translation system using [35S]methionine, [3H]leucine, [3H]threonine, [3H]proline, or [3H]serine as radiolabel and the translation products were analyzed by gel electrophoresis and fluorography before and after immunoprecipitation with the antiserum. A labeled product of molecular weight 60,000 was present in the immunoprecipitates obtained in the absence but not in the presence of the unlabeled competitor deglycosylated mucin. It is concluded that the primary translation product of the bovine submaxillary gland gene is a 60,000-dalton protein and that the monomer subunit of the mucin is about 170,000. Thus, in the native state the mucin consists of several self-associating subunits.     

Characterization of the secretory proteins of rat preputial gland in relation to urinary proteins

The water-soluble proteins of the rat preputial gland secretion were characterized in native and SDS-treated form on polyacrylamide gel electrophoresis. Nine major proteins were present in the secretion. One protein was a glycoprotein of molecular weight greater than 200,000 with beta-glucuronidase activity, and the other eight proteins had a molecular weight of 17,000, but with different charges. Acid phosphatase and arylsulphatase activities were present in the secretion in minor amounts. The isoelectric points of the secretory proteins ranged from 8.5 to 5.3; none of the proteins were lipoproteins, and there were no sex differences. The male and female rat urinary proteins were also characterized electrophoretically. The male rats had two different protein patterns, probably genetically determined. The female rats showed basically one urinary protein pattern, but their urines were frequently mixed with the preputial gland secretory proteins, which most likely played a part in the chemical communication. The mixing could not be correlated to daytime or estrous cycle.     

Purifications of Rat Adrenal Dopamine-β-Hydroxylase: Immunological Analysis of Its Soluble and Membrane-Bound Forms with the Use of an Antibody Raised Against the Soluble Form

Soluble and membrane-bound dopamine-beta-hydroxylases (sDBH and mDBH, respectively) from rat adrenal glands have been purified through concanavalin A-Sepharose chromatography, gel filtration, and ion-exchange high-performance chromatographies. Both sDBH and mDBH were composed by four subunits of apparent molecular weight of 75,000 and showed a native molecular weight of 300,000. This procedure has not allowed us to obtain a sufficient amount of enzyme to immunize a rabbit. A second, more rapid procedure was designed to isolate sDBH, including concanavalin A-Sepharose chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was raised against this purified protein. The specificity of the antiserum was demonstrated by neutralization of rat adrenal gland DBH activity, labeling of rat adrenal medulla on histological sections, and, after Western blot, labeling of the 75,000-molecular-weight band in the different fractions associated with DBH activity during purification. The antiserum had a higher affinity for the sDBH denatured by sodium dodecyl sulfate than for the native protein. It had a higher affinity for sDBH than for mDBH. These results strongly suggest the presence of specific hydrophilic epitopes on the sDBH, revealing structural differences between the two hydroxylase forms. Two protein bands were stained on Western blots of crude rat adrenal gland extract. One band had an apparent molecular weight of 75,000, and the other of 82,000. Our results showed that the two proteins contained similar epitopes, an observation suggesting a close structural relationship. The higher-molecular-weight protein could be the 75,000 protein before covalent modifications and cleavage.     

Studies on some aspects of peroxidase from submaxillary gland

A peroxidase has been purified 25- to 30-fold over crude homogenate from goat submaxillary gland, which shows a single band of protein on polyacrylamide gel electrophoresis at four different pH values (4.6–10.0). A molecular weight (M_r) of approximately 2 × 10⁴ per heme binding site has been found. The molecular weight of the enzyme determined by Sephadex-gel filtration method, appeared to be 4 × 10⁴. The sedimentation pattern of the purified enzyme shows a symmetrical peak, although there was evidence of some small heterogenous material near the meniscus. The sedimentation coefficient of the enzyme (s^o 20^wat 0.4% of the enzyme concentration) was found to be 4.18, which indicates the molecular weight of the enzyme to be approximately 6 × 10⁴.     

Binding of ferulic acid to cell walls by peroxidases of Pinus elliottii

Lignin is formed abundantly in the maturing walls of slash pine cambial cells, but very little in slash pine callus cell walls. Peroxidases removed from the cytoplasm of callus or cambial cells with phosphate buffer (soluble peroxidase), from the walls with NACl (ionically bound peroxidase), and from the walls with cellulase (covalently bound peroxidase) differed in their capacity to catalyze bond formation between carbohydrate and ferulic acid or its condensation products. Bond formation per unit of enzyme was highest in the peroxidases of cambium, especially in those attached ionically or covalently to the cell walls. The wall-bound peroxidases also catalyzed the strongest linkages between lignin monomers and carbohydrates as estimated by their resistance to hydrolysis by NaOH.     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis