Chi P. Cheung

Analysis of inorganic pyrophosphate at the picomole level.

Abstract A radiometric determination of inorganic pyrophosphate is described in this communication. Excess uridine 5′-diphospho[ 14 C]glucose is used to react with inorganic pyrophosphate in a reaction catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. This reaction liberates [ 14 C]glucose 1-phosphate, which is then further converted to 6-phospho[ 14 C]gluconic acid in the presence of phosphoglucose mutase and glucose 6-phosphate dehydrogenase. The reaction product, 6-phospho[ 14 C]gluconic acid, is separated from uridine diphospho[ 14 C]glucose by the addition of activated charcoal, which adsorbs uridine diphospho[ 14 C]glucose preferentially. The sensitivity of the assay for inorganic pyrophosphate is 20 pmol.

Regulation of RNA synthesis in early germination of isolated wheat (Triticum aestivum L) embryo

THE change from dormancy to germination of seeds requires water and an appropriate temperature. Wheat embryo germination is characterised by an initial increase in fresh weight, followed by a 5-h lag1. Although RNA synthesis is one of the earliest biologically measurable activities in wheat embryo germination2,3, its mode of regulation has not been established. Reports3–6 have suggested that the rate of synthesis does not change during the first 6 h of germination. But no adjustment was made for the specific activities of ribonucleoside triphosphate, UTP. This would have led to a misinterpretation of incorporation data when there was either a change in the concentration of the nucleotide or a difference in the rate of phosphorylation from nucleoside to nucleotide as germination progressed. To investigate further whether there is a change in the rate of RNA synthesis in the germination phase, we have used recentlydeveloped enzyme assays7–9 to measure picomole changes in purine pyrimidine ribonucleotide levels. We found a threefold increase in the rate of RNA synthesis of wheat embryos germinated for 40 min–5.5 h. We used the reaction catalysed by uridine 5′-diphosphoglucose pyrophosphorylase to determine the content of UTP. The key step in this analysis is the selective adsorption of the reaction product, UDP-14C-glucose, on to activated charcoal in the presence of 0.8 M Trizma base9.

A rapid quantitative method for measuring UTP, UDP, and UMP in the picomole range.

Abstract A procedure for the determination of picomole amounts of uracil nucleotides is described. The key reaction is the condensation of UTP and [ 14 C]glucose 1-phosphate catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase yielding UDP-[ 14 C]glucose. The product is determined by selective adsorption onto charcoal in the presence of 0.8 m Trizma Base. UDP is measured as UTP after its conversion in an incubation with excess ATP and nucleoside diphosphate kinase. Similarly, UMP is analyzed after it is converted to UDP by nucleoside monophosphate kinase. The uracil nucleotide content of germinated wheat embryos had been determined with this method.

The reversal of inhibition of protein synthesis by double-stranded RNA in lysed rabbit reticulocytes with fructose 6-phosphate.

Abstract Fructose 6-phosphate (1.4 mM – 3.0 mM) effectively prevents the inhibition of protein synthesis in unfractionated rabbit reticulocyte lysates by the presence of double-stranded RNA (poly rI:poly rC, 1 μg/ml). Glucose 6-phosphate, but not fructose 1,6-diphosphate, is equally as effective as fructose 6-phosphate. The data suggest that fructose 6-phosphate prevents the formation of a protein synthesis inhibitor induced by double-stranded RNA.

Inhibition of protein synthesis by glucose 6-phosphate and fructose 1,6-diphosphate in lysed rabbit reticulocytes and the reversal of inhibition by NAD+

Abstract Glucose 6-phosphate and fructose 1,6-diphosphate inhibit protein synthesis when added to lysed rabbit reticulocytes. Protein synthesis is inhibited 47% with 6 mM fructose 1,6-diphosphate and 86% with 6 mM glucose 6-phosphate. With 0.125 mM NAD + , the inhibitory effect of glucose 6-phosphate and fructose 1,6-diphosphate becomes stimulatory. The stimulation of protein synthesis in those assays with NAD + and the phosphorylated sugars is 50% above those assays that contain NAD + alone. The inhibition of protein synthesis by glucose 6-phosphate and the reversal of this inhibition by NAD + occurs at a step before the synthesis of the initial dipeptide, methionyl-valine. These data illustrate the importance of NAD + and the activation of glycolysis in regulating protein synthesis in lysed rabbit reticulocytes.

Analysis of NAD+ in picomole amounts with sodium [32P]pyrophosphate and NAD-pyrophosphorylase

Abstract A new method is described for the determination of NAD + in picomole amounts. An enzymatic coupling system of NAD-pyrophosphorylase and hexokinase is used to convert sodium [ 32 P]pyrophosphate and NAD + to [ 32 P]ADP, glucose 6-[ 32 P]phosphate, and NMN. The key step in this analysis is the selective adsorption of the reaction product [ 32 P]ADP, onto activated charcoal with a solution of 1 m K 2 HPO 4 :10% trichloroacetic acid (1:3, v/v, pH 2). The range of concentrations of NAD + that can be measured is 1–200 pmol. The simplicity of the method allows as many as 180 samples to be assayed in 4–5 h. This procedure has been used to quantitate NAD + in crude extracts of germinating wheat embryos.

Analysis of glucose 1-phosphate at the picomole level with [14C]UTP and uridine 5′-diphosphoglucose pyrophosphorylase☆

A new sensitive method is described for glucose 1-phosphate analysis. The key reaction is the pyrophosphorolysis of UDP-glucose catalyzed by uridine 5′-diphosphoglucose pyrophosphorylase. The reaction product, [14C]UDP-glucose, is separated from [14C]UTP by adsorbing [14C]UTP selectively onto polyethyleneimine cellulose or by separating both labeled compounds on one-dimensional polyethyleneimine thin-layer chromatograms. The sensitivity of the method for glucose 1-phosphate analysis is 5 pmol. The method has been successfully employed to monitor the level of glucose 1-phosphate in early germination of wheat embryos.

REGULATION OF PROTEIN SYNTHESIS: ROLE OF NAD+ AND SUGAR PHOSPHATES IN LYSED RABBIT RETICULOCYTES

This chapter describes the regulation of protein synthesis and role of NAD + and sugar phosphates in lysed rabbit reticulocytes. NAD + , NAD + , analogs, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and cAMP have been shown to stimulate and/or inhibit protein synthesis in lysed rabbit reticulocytes. NAD + and NAD + analogs can replace the energy requirements for CP/CPK and PEP/PK in cell-free synthesizing systems of lysed rabbit reticulocytes. NAD + at 160 μM stimulates the initiation step of the protein synthetic process as determined by the transfer of formyl-methionine from f[ 35 S]Met-tRNA met into polypeptide and the accumulation of methionyl-valine in the presence of pactamycin. The effect of NAD + on protein synthesis is closely correlated with the activation of glycolysis. Activation of glycolysis by NAD + generates ATP, thereby maintaining a high energy charge. The conversion of glycolytic intermediates to lactate may trigger a signal that stimulates the initiation of protein synthesis. The addition of hexose phosphates and NAD + stimulates protein synthesis to a greater extent than that observed with NAD + alone. Fructose-6-phosphate prevents the formation of a protein synthesis inhibitor induced by double-stranded RNA.