Purification and characterization of a high molecular weight ribonuclease from human milk

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...

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Bibliographic Details
Published in:The Journal of biological chemistry 1993-02, Vol.268 (6), p.4181-4187
Main Authors: HEMAVATHI RAMASWAMY, BRAHMENDRA SWAMY, C. V, RAMACHANDRA DAS, M
Format: Article
Language:English
Subjects:
Amino Acids - analysis
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Cations
Chromatography, Liquid
Electrophoresis, Polyacrylamide Gel
Enzyme Stability
Enzymes and enzyme inhibitors
Female
Fundamental and applied biological sciences. Psychology
Hot Temperature
Humans
Hydrogen-Ion Concentration
Hydrolases
Kinetics
Milk, Human - enzymology
Molecular Weight
Ribonucleases - chemistry
Ribonucleases - isolation & purification
Ribonucleases - metabolism
RNA - metabolism
Substrate Specificity
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)53595-x