Substrate-leash amplification with ribonuclease S-peptide and S-protein.
The S-peptide and S-protein fragments of ribonuclease S (RNase S, no EC no. assigned) have been immobilized onto separate Sepharose gels via a "leash" of polycytidylic acid substrate. Each of these gels releases its RNase fragment when treated with the complementary enzyme fragment or with RNase A (EC 3.1.27.5), and the released fragments recombine to give RNase S activity. Thus this system provides substrate-leash amplification (SLA), such that more enzymatic activity is eluted from the system than is applied. For example, 100 pg of RNase applied to the S-peptide gel is amplified by 1.9 X 10(4) to the equivalent of 1.9 micrograms of activity in 20 h, when followed by combination of the released S-peptide with excess S-protein. We also tested a three-stage amplification system, with a pair of S-peptide and S-protein gels at each stage. In this system the cumulative amplification of the initial 1-ng dose of RNase A is 4.9, 52, and 25-fold after each stage, respectively. Only 2 mg of each SLA gel is used per stage in these experiments, reflecting the magnitude of their production of RNase S activity.
D000818:Animals; D002417:Cattle; D004800:Enzymes, Immobilized / Q000378:metabolism*; D008722:Methods; D010446:Peptide Fragments / Q000378:metabolism*; D011066:Poly C / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; C023560:ribonuclease S-peptide; D011066:Poly C; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S