Milacemide metabolism in rat liver and brain slices by solids NMR.

The metabolism of 13C- and 15N-labeled milacemide, 2-(pentylamino)-acetamide, has been studied in rat liver and brain slices using solid-state NMR. This analysis is fast and efficient and can be used to monitor both major and minor metabolic pathways in mammalian tissue culture. The NMR work reported herein involves both conventional cross-polarization magic-angle spinning 13C and 15N NMR spectra and rotational-echo double resonance 13C-15N experiments. The latter measure quantitatively the breaking of isotopically labeled carbon-nitrogen chemical bonds. Our results, which are consistent with suggestions from previous metabolic studies, show that the first step in the breakdown of milacemide is the breaking of the pentylamine nitrogen bond to yield pentanoic acid and glycinamide. Total incorporation of 15N label from the resulting glycinamide fragment is comparable in rat liver and brain. In both tissues, considerably more of the 15N label from glycinamide is incorporated than the corresponding 13C label. Differences between the liver and brain tissue are also observed, with more synthesis incorporating the 13C labels taking place in the liver.

Solid-state NMR observation of cysteine and lysine Michael adducts of inactivated estradiol dehydrogenase.

The inactivation of estradiol dehydrogenase by enzyme-generated 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one is accompanied by the formation of a lysine enaminone. The experiments leading to this conclusion involved degradation of the inactivated enzyme with Pronase and subsequent analysis by solution-state 13C NMR. The present paper reports solid-state 13C NMR experiments on lyophilized intact inactivated enzyme which are free from problems due to Pronase digestion. These experiments combine conventional cross-polarization and magic-angle spinning with selective irradiation of resonances arising from a 13C double label in the steroid. Magnetization transfer between neighboring 13C nuclei is used to simplify the spectra and to identify peaks due to label. The formation of cysteine and lysine Michael adducts of the enzyme is established by comparisons with chemical shifts of solid model adducts.

Disparate molecular dynamics of plasmenylcholine and phosphatidylcholine bilayers.

The molecular dynamics of binary dispersions of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol were quantified by electron spin resonance (ESR) and deuterium magnetic resonance (2H NMR) spectroscopy. The order parameter of both 5-doxylstearate (5DS) and 16-doxylstearate (16DS) was larger in vesicles comprised of plasmenylcholine in comparison to phosphatidylcholine at all temperatures studied (e.g., S = 0.592 vs. 0.487 for 5DS and 0.107 vs. 0.099 for 16DS, respectively, at 38 degrees C). Similarly, the order parameter of plasmenylcholine vesicles was larger than that of phosphatidylcholine vesicles utilizing either spin-labeled phosphatidylcholine or spin-labeled plasmenylcholine as probes of molecular motion. The ratio of the low-field to the midfield peak height in ESR spectra of 16-doxylstearate containing moieties (i.e., spin-labeled plasmenylcholine and phosphatidylcholine) was lower in plasmenylcholine vesicles (0.93 +/- 0.01) in comparison to phosphatidylcholine vesicles (1.03 +/- 0.01). 2H NMR spectroscopy demonstrated that the order parameter of plasmenylcholine was greater than that of phosphatidylcholine for one of the two diastereotopic deuterons located at the C-2 carbon of the sn-2 fatty acyl chain. The spin-lattice relaxation times for deuterated plasmenylcholine and phosphatidylcholine in binary mixtures containing 0-50 mol % cholesterol varied nonmonotonically as a function of cholesterol concentration and were different for each phospholipid subclass. Taken together, the results indicate that the vinyl ether linkage in the proximal portion of the sn-1 aliphatic chain of plasmenylcholine has substantial effects on the molecular dynamics of membrane bilayers both locally and at sites spatially distant from the covalent alteration.