title: Purification and characterization of a high molecular weight ribonuclease from human milk.
abstract: We have purified a high molecular weight ribonuclease (hmRNase) from human milk by a two-step column chromatographic procedure and characterized the enzyme. The molecular mass of hmRNase is 80 kDa as determined from SDS-polyacrylamide gel electrophoresis. The pH optimum of the enzyme is in the range of 7.5-8.0, similar to other secretory RNases. hmRNase is pyrimidine-specific and cleaves the phosphodiester bond 3' to a pyrimidine residue. It selectively degrades the pyrimidine strand in poly(rA):poly(rU) and poly(dA):poly(rU) double stranded substrates. The extent of degradation for naturally occurring RNAs vary in the order tRNA < rRNA < mRNA at low enzyme concentrations. hmRNase shows allosteric behavior with positive cooperativity in its reaction on polynucleotide substrates. The activity of the enzyme is enhanced in the presence of monoribonucleotides. Antiserum obtained against purified hmRNase did not cross-react with low molecular weight RNase which is also present in milk. In addition, an immunologically cross-reacting species could not be detected in the serum, suggesting the origin of hmRNase in the mammary gland but not blood.
authors.affiliation: Center for Cellular and Molecular Biology, Hyderabad, India.
authors.forename: H
authors.initials: H
authors.lastname: Ramaswamy
authors.forename: C V
authors.initials: CV
authors.lastname: Swamy
authors.forename: M R
authors.initials: MR
authors.lastname: Das
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002412:Cations; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005260:Female; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008895:Milk, Human / Q000201:enzymology*; D008970:Molecular Weight; D012313:RNA / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D002412:Cations; D012313:RNA; D012260:Ribonucleases