Human non-secretory ribonucleases. I. Purification, peptide mapping and lectin blotting analysis of the kidney, liver and spleen enzymes.
Human non-secretory neutral ribonucleases (RNases) from kidney, liver and spleen have been purified and characterized. SDS-PAGE indicates that all three RNases are highly purified and have apparent mol. wts of 17-18 kDa. Kinetic analysis indicates that all three RNases have a broad pH optimum centred around 6.5, and all three have similar substrate specificities with significant preference for RNA and poly(U) when compared to poly(C), poly(A) and poly(G). All of the above data, as well as immunoblotting data using three polyclonal antibodies (anti-human liver RNase, anti-human pancreatic RNase, anti-human eosinophil-derived neurotoxin), indicate that the three proteins are highly purified and are non-secretory RNases (IIN). Further characterization by cyanogen bromide peptide mapping and extensive lectin blotting indicated no significant differences between the three human RNases. All three RNases appear to have very similar, if not identical, protein backbones and all three are glycoproteins which are recognized by lectins with specificity for GlcNAc, Fuc and, to a lesser extent, with specificity for Gal beta(1-4)GlcNAc. No significant tissue-specific differences were found among the three human non-secretory RNases.
Lawrence
C W
CW
Department of Chemistry, Lehigh University, Bethelehem, PA 18015.
Little
P A
PA
Little
B W
BW
Miller
M J
MJ
Bazel
S
S
Alhadeff
J A
JA
D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D037102:Lectins; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping; D012260:Ribonucleases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology*; D013379:Substrate Specificity
D006023:Glycoproteins; D037102:Lectins; D012260:Ribonucleases