Mode of orthophosphate uptake and ATP labeling by mammalian cells

Incubation of HeLa cells with [³²P]orthophosphate results in more rapid labeling of the γ-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4–7.8. At lower pH, 6.8–7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

Enhanced nucleotide analysis enables the quantification of deoxynucleotides in plants and algae revealing connections between nucleoside and deoxynucleoside metabolism

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.

A new method for the quantification of (deoxy)ribonucleotides and nucleosides enables an in-depth analysis of the nucleotide metabolism in plants.     

Nitrate induces enzymes of the mannitol cycle and suppresses versicolorin synthesis inAspergillus parasiticus

Addition of sodium nitrate to growing cultures ofAspergillus parasiticus (ATCC 36537) induces the synthesis of enzymes involved in nitrate assimilation (NO ₃ ^– reductase), of enzymes in the pentose pathway (glucose-6-phosphate dehydrogenase), and of enzymes in the mannitol cycle (mannitol- and mannitol-1-phosphate dehydrogenases). Addition of NO ₃ ^– also causes a dose-dependent suppression of synthesis of the polyketide secondary metabolite, versicolorin A. We suggest that in the presence of NO ₃ ^– plus peptone, the cytoplasmic NADPH/NADP ratio may be elevated, resulting in increased conversion of malonyl coenzyme A to fatty acid rather than to polyketide.     

A proposed role of superoxide anion as a biological nucleophile in the deesterification of phospholipids

Potassium superoxide dissolved in dry dimethyl sulfoxide effects rapid deesterification of ethyl hexadecanoate and of dilauroyl phosphatidyl choline. The reaction with ethyl hexadecanoate is reversible, having an apparent equilibrium constant of 0.4. It is proposed that some of the deleterious effects on biological membranes which have been atributed to oxidation by superoxide may actually be the result of deesterification by superoxide acting as a nucleophile.