Denaturation of thymidylate synthetase from amethopterin-resistant Lactobacillus casei.
The effects of various concentrations of urea and guanidine hydrochloride on enzyme activity and on subunit association were determined. Incubation of thymidylate synthetase with buffered solutions of 3M to 3.5M guanidine hydrochloride or 5 M to 6 M urea resulted in the loss of about 90% of the enzyme activity. Under these denaturing conditions a red shift of the fluorescence emission maximum from 340 nm to 351 nm was observed together with a significant decrease in the relative fluorescence intensity of the protein. Studies at both 4 degrees C and 25 degrees C indicated that the enzyme was in the dimer form in 2 M guanidine hydrochloride but was dissociated into monomers in concentrations of this denaturant of 3 M and above. Although only monomeric species were evident at 4 degrees C in 6 M urea, at 25 25 degrees C this denaturant caused protein aggregation which increased with decreasing phosphate buffer concentration. Enzyme (5 mg/ml) in 0.5 M potassium phosphate buffer, pH 6.8, containing 4 M guanidine hydrochloride gave a minimum S20, w value of 1.22S at 25 degrees C. Sedimentation behavior of the native enzyme in the range of 5 to 20 mg/ml was only slightly concentration-dependent (4.28 S to 4.86 S) but extensive aggregation occurred above 20 mg/ml.
D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004352:Drug Resistance, Microbial; D006146:Guanidines / Q000494:pharmacology; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D046911:Macromolecular Substances; D008727:Methotrexate / Q000494:pharmacology; D008780:Methyltransferases / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013940:Thymidylate Synthase / Q000378:metabolism*; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D046911:Macromolecular Substances; D014508:Urea; D008780:Methyltransferases; D013940:Thymidylate Synthase; D008727:Methotrexate