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.
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P K
PK
Centre for Cellular and Molecular Biology, Hyderabad, India.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003208:Concanavalin A; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D005260:Female; D005779:Immunodiffusion; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D007765:Lacrimal Apparatus / Q000201:enzymology*; D007774:Lactation; D008297:Male; D008321:Mammary Glands, Animal / Q000201:enzymology; D051379:Mice; D008970:Molecular Weight; D009928:Organ Specificity; D010413:Penis / Q000201:enzymology; D010544:Peroxidases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013363:Submandibular Gland / Q000201:enzymology; D014599:Uterus / Q000201:enzymology
D007527:Isoenzymes; D003208:Concanavalin A; D010544:Peroxidases