One-carbon units are essential to a variety of anabolic processes which yield necessary cellular components including purines, pyrimidines, amino acids, and lipids. Serine hydroxymethyltransferase (SHMT) is the major provider of one-carbon units in the cell. The other product of this reaction is glycine. Both of these metabolites are required in de novo purine biosynthesis. In Saccharomyces cerevisiae, mitochondrial and cytoplasmic SHMT isozymes are encoded by distinct nuclear genes (SHM1 and SHM2). Molecular genetic analyses have begun to define the roles of these two isozymes in folate-mediated one-carbon metabolism [McNeil, J. B., et al. (1996) Genetics 142, 371-381]. In our study, the SHM1 and SHM2 genes were disrupted singly and in combination to investigate the contributions of the two SHMT isozymes to the production of glycine and one-carbon units required in purine biosynthesis. Cell subfractionation experiments indicated that while only 5% of total activity was localized in the mitochondria, the specific activity in that compartment was much higher than in the cytoplasm. Growth and 13C NMR experiments indicate that the two isozymes function in different directions, depending on the nutritional conditions of the cell. When yeast was grown on serine as the primary one-carbon source, the cytoplasmic isozyme was the main provider of glycine and one-carbon groups for purine synthesis. When grown on glycine, the mitochondrial SHMT was the predominant isozyme catalyzing the synthesis of serine from glycine and one-carbon units. However, when both serine and glycine were present, the mitochondrial SHMT made a significant contribution of one-carbon units, but not glycine, for purine synthesis. Finally, NMR data are presented that suggest the existence of at least two sites of de novo purine biosynthesis in growing yeast cells, each being fed by distinct pools of precursors.
Liposome-based nanoSized Particles with Incorporated Nitroxides, or nanoSPINs, were designed for EPR applications as pH probes in biological systems. Phospholipid membrane of the liposomes with incorporated gramicidin A showed selective permeability to a small analyte, H + , while protecting entrapped sensing nitroxide from biological reductants. An application of the pH-sensitive nanoSPIN in an ischemia model in rat heart homogenate allows for monitoring ischemia-induced acidosis while protecting encapsulated nitroxide against bioreduction.
Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases sub-nanomolar physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescent approach for direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphopshate to guanosine 3’, 5’-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases about 200 times when NO binds with sGC, and in so doing provides a sensor for nitric oxide. Luminescent detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from ATP-dependent luciferin-luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurements of NO generated by nitric oxide donor (PAPA-NONOate), nitric oxide synthase and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.
A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin–luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 102–104 cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM).
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