Interaction of human placental ribonuclease with placental ribonuclease inhibitor.
The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed previously, nor have any placental RNases been identified. In the present work, the major human placental RNase (PR) was purified to homogeneity by a five-step procedure and was obtained in a yield of 110 micrograms/kg of tissue. The placental content of angiogenin was also examined and was found to be at least 10-fold lower than that of PR. On the basis of its amino acid composition, amino-terminal sequence, and catalytic properties, PR appears to be identical with an RNase previously isolated from eosinophils (eosinophil-derived neurotoxin), liver, and urine. The apparent second-order rate constant of association for the PR.PRI complex, measured by examining the competition between PR and angiogenin for PRI, is 1.9 X 10(8) M-1 s-1. The rate constant for dissociation of the complex, determined by HPLC measurement of the rate of release of PR from its complex with PRI in the presence of a scavenger for free PRI, is 1.8 X 10(-7) s-1. Thus the Ki value for the PR.PRI complex is 9 X 10(-16) M, similar to that obtained with angiogenin, and 40-fold lower than that measured with RNase A. Complex formation causes a small red shift in the protein fluorescence emission spectrum, with no significant change in overall intensity. The fluorescence quantum yield of PR and the Stern-Volmer constant for fluorescence quenching by acrylamide are both high, possibly due to the presence of an unusual posttranslationally modified tryptophan residue at position 7 in the primary sequence.
Shapiro
R
R
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115.
Vallee
B L
BL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015226:Dinucleoside Phosphates / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D010926:Placental Hormones / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D015226:Dinucleoside Phosphates; D010926:Placental Hormones; D011506:Proteins; C022052:placental ribonuclease inhibitor; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic