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.
Highlights
RNases have been recognized toconstitute a superfamily, in which several diverse proteins such as angiogenin and eosinophil cationicproteinare shown to share present in milk
Two peaks of RNase activity were obtained, and the fractions corresponding to the major peak eluting at 0.7-1.0 M NaCl were pooled, concentrated, and dialyzed in 10 mM Tris-HC1 and 1mM phenylmethylsulfonyl fluoride (PMSF) (Trisbuffer)
Identification of a High Molecular Weight Ribonuclease-In human milk there are at least two proteins having RNase activity; one of them has a molecular mass of 80 kDa and the minorspotcorresponding to cytosine, perhaps due to the other 14 kDa(Fig. 1)
Summary
RNases have been recognized toconstitute a superfamily, in which several diverse proteins such as angiogenin and eosinophil cationicproteinare shown to share present in milk. The zymogram of human milk plasma revealed two bands with RNase activity (Fig. 1, lane I ) corresponding to molecular masses of 80 and 14 kDa, respectively.
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call