Hypersensitive substrate for ribonucleases.
A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 3.6 x 10(7)M(-1)s(-1). Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3. 3 x 10(2)M(-1)s(-1). This value is >10-fold larger than that for other known substrates of angio-genin. With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.
Kelemen
B R
BR
Department of Biochemistry, University of Wisconsin-Madison, WI 53706, USA.
Klink
T A
TA
Behlke
M A
MA
Eubanks
S R
SR
Leland
P A
PA
Raines
R T
RT
D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011506:Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013816:Thermodynamics; D014542:Uridine Monophosphate / Q000378:metabolism / Q000494:pharmacology
C114557:5-carboxytetramethylrhodamine succinimidyl ester; D003839:Deoxyadenosines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011506:Proteins; D011743:Pyrimidines; D012235:Rhodamines; C024098:6-carboxyfluorescein; D000244:Adenosine Diphosphate; D014542:Uridine Monophosphate; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; C030986:pyrimidine