Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions.
Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.
Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions. Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.