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Biomedical subjects

R B Dunlap

Publications and source records attributed to R B Dunlap.

At least 19 recordsLinked to original sources
PubMed1979

Modification of lysyl residues of dihydrofolate reductase with 2,4-pentanedione.

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) from an amethopterin-resistant strain of Lactobacillus casei was inactivated by 2,4-pentanedione. The inactivation appears to be due to the specific interaction of 2,4-pentanedione with lysyl residues. Inactivation is concomitant with with the modification of three lysyl residues. Both NADPH and dihydrofolate protect the enzyme against inactivation, suggesting that the critical residue(s) lies at or near their binding sites. Unlike native dihydrofolate reductase, 2,4-pentanedione-modified enzyme does not form binary complexes with either NADPH, dihydrofolate or amethopterin which are stable to gel filtration. Treatment of the modified enzyme with nucleophilic reagents such as hydroxylamine, failed to promote reactivation of the enzyme. Reactivation was achieved following gel filtration at pH 6.0 and was found to be dependent on the degree to which the enzyme was inactivated.

Arginine↗
PubMed1979

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.

Chromatography, Gel↗
PubMed1978

Interactions between tetrahydrofolate and palladium(II) complexes.

Interactions between folate derivatives and palladium(II) complexes are monitored in 1 : 1 molar ratio mixtures by circular dichroic spectra. The results are consistent with the following conclusions, i.e., tetrahydrofolate forms a chelate complex with palladium(II) through nitrogens 5 and 10 which is characterized by a unique circular dichroic spectrum. Mixtures of pallodium (II) complexes and dihydrofolate on methylenetetrahydrofolate could not be expected to, nor do they, give rise to similar circualr dichroic spectra for the following reasons: (1) The N5, N10 chelation site of tetrahydrofolate is blocked in methylenetetrahydrofolate, so coordination of a palladium(II) species must occur at some secondary site. (2) The N5, N10 chelation site is available but carbon six of the pteridine ring system is no longer asymmetric in dihydrofolate. Mixtures of dihdrofolate and palladium(II) complexes have no measurable circular dichroic spectra under the experimental conditions used.

Binding Sites↗