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.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26±Cys84, Cys40±Cys95, Cys58±Cys110, and Cys65±Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26±Cys84 and Cys58±Cys110) enhance stability more than do the two embedded ones (Cys40±Cys95 and Cys65±Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 8C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of k cat /K m that are 2-to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.Keywords: conformational stability; differential scanning calorimetry; disulfide bond; enzyme; ribonuclease A.A polypeptide chain can adopt many conformations. Yet, the sequence of its amino-acid residues directs folding to a particular native state [1]. The loss of conformational entropy associated with folding destabilizes the native conformation. This destabilization is overcome by the hydrophobic effect, hydrogen bonds, other noncovalent interactions, and (for many proteins) disulfide bonds [2].Bovine pancreatic ribonuclease A (RNase A; EC 3.1.27.5 [3,4]) provides a superb template with which to dissect the contribution of disulfide bonds to conformational stability. RNase A consists of 124 amino-acid residues and contains four intrachain disulfide bonds (Cys26±Cys84, Cys40±Cys95, Cys58±Cys110, and Cys65±Cys72; Fig. 1). The four disulfide bonds are conserved in all 40 of the known sequences of homologous mammalian pancreatic ribonucleases [5]. Two disulfide bonds (Cys40±Cys95 and Cys65±Cys72) link together surface loops, and two link an a-helix to a b-sheet in the protein core (Cys26±Cys84 and Cys58±Cys110). Three disulfide bonds enclose a loop of similar size (Cys26±Cys84, Cys40±Cys95 and Cys58±Cys110; h 59, 56, and 53, respectively), and the other disulfide bond (Cys65±Cys72; h 8) encloses a smaller loop. In previous work, the cystines of RNase A were replaced with pairs of serine ...

Onconase™, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/ G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. The free energy difference between the native and unfolded states of a protein is small-typically 5-15 kcal/mol (1, 2). In their native state, many proteins are less susceptible to proteolytic degradation than when unfolded (3). In the unfolded state, the steric protection of peptide bonds provided by the compact native state is lost (4). For example, bovine pancreatic ribonuclease A (RNase A) 1 (EC 3.1.27.5 (5, 6)) is degraded more readily by proteases in the presence of denaturants or elevated temperatures (7-10) and less readily when glycosylated (11).The rates of intracellular protein turnover vary by 10 3 -fold (12). Apparently, some proteins are better able to thwart the proteolytic machinery (13,14). These proteins remain intact and retain activity longer within the cell. A correlation between the conformational stability of a protein and its catabolism was reported over 20 years ago (15). Since then, studies using unrelated proteins have either supported (16) or contradicted (14, 17) this correlation. Drawing conclusions from these studies is problematic because the proteins were divergent in characteristics that have been implicated in metabolic turnover. In contrast, variants of a single protein, the N-terminal domain of the repressor protein from bacteriophage , have been used to demonstrate a definite link between conformational stability and metabolic turnover (18). More recently, the conformational stability of bovine pancreatic trypsin inhibitor variants was shown to correlate with the yield of intact protein produced in a heterologous system (19).RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which ...

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ATP depletion and ADP formation are generic detection methods used for the identification of kinase and other ATP-utilizing enzyme inhibitors in high-throughput screening campaigns. However, the most widely used nucleotide detection approaches require high ATP consumption rates or involve the use of coupling enzymes, which can complicate the selection of lead compounds. As an alternative, we have developed the Transcreener (BellBrook Labs, Madison, WI) platform, which relies on the direct immunodetection of nucleotides. Here we describe the development of antibodies with >100-fold selectivity for ADP versus ATP, which enable robust detection of initial velocity rates (Z' > 0.7 at 10% substrate consumption) at ATP concentrations ranging from 0.1 microM to 1,000 microM in a competitive fluorescence polarization (FP) immunoassay. Competitive binding experiments indicate similar affinities for other nucleotide diphosphates, including 2' -deoxy ADP, GDP, and UDP. The antibody-tracer complex and the red-shifted, ratiometric FP signal are stable for at least 24 h at room temperature, providing suitable conditions for high-throughput screening. A method for calculating a kinase ATP Km with this FP immunoassay is also presented. The Transcreener ADP assay provides a simple, generic assay platform for inhibitor screening and selectivity profiling that can be used for any ADP-generating enzyme.