No abstract

Spin-labeled copolymers of 4-thiouridine and uridine [(ts4UU)] that contain various amounts of spin label (e) were synthesized by either (i) chemical alkylation of the 4-thiouridine-uridine copolymers (s4U,U). prepared by copolymerizing 4-thiouridine 5'-diphosphate (s4UDP) and UDP or (ii) copolymerization of spin-labeled S4UDP with UDP using polynucleotide phosphorylase. The effect of (s4U,U). and (tS4UU). on avian myeloblastosis virus (AMV) RNA-dependent DNA polymerase (RNA-dependent DNA nucleotidyltransferase, EC 2.7.7.7; reverse transcriptase) was studied to determine whether the presence of potentially reactive thiol groups or spin labels enhances the inhibitory properties of the copolymers as compared to (U)3. Inhibition by (s4U,U) gradually increases as the percentage of thiolation increases. Enhanced inhibition by (s4U,U). appears to be due to the interaction of the thiol groups of (s4U,U). with the thiol group(s) of the polymerase, because inhibition by (s4U,U). (8% thiolated) in the presence of dithiotreitol resembles that by (U)L. In contrast, inhibition by (ts4UU). containing 3% spin label resembles that by (U)L; however, increasing the spin label to 6% or 12% results in enhanced inhibition by (4s4U,U). as compared to that by (U)3, and dithiothreitol has no effect on enhanced inhibition by (t UU)3.These results suggest that the mechanism of inhibition observed with (ts4U,U). with a es4U:U ratio > 1:33 differs from the mechanism for (s4U,U). and involves complex formation between the spin label and the essential Zn2+ of RNA-dependent DNA polymerase.

Modification of the single cysteine residue of streptococcal dihydrofolate reducatase with 5,5'-dithiobis (2-nitrobenzoic acid) results in virtually complete inactivation of the enzyme. Reduction of the enzyme-S-nitrobenzoate mixed disulfide with dithiothreitol indicates that 1.0 cysteine has been modified and approximately 70 percent of the original enzyme activity restored. Circular dichroic and fluorescence studies suggest that a localized conformational change involving aromatic residues, possibly tryptophan, has occurred following modification. In addition, nearly stoichiometric amounts of p-hydroxymercuribenzoate lead to complete inactivation of the reducatas. It is suggested that the S-nitrobenzoate moiety bound to the cysteine residue of the enzyme may perturb one or more aromatic side chains and lead to a distortion of the hydrophobic substrate binding site.

The sensitive biological assay for reverse transcriptase was used to monitor potential perturbation effects of spin labels covalently bound to various nucleic acids or nucleic acid analogs to the extent of about one label per 100 residues. The inhibitory properties of the spin labeled and unlabeled biopolymers were compared for evaluating possible interference of the reporter group in protein-nucleic acid interaction studies. The amount of inhibitor required for 50% inhibition (ED50) was determined for the