Phyllis R. Brown

The rapid separation of nucleotides in cell extracts using high-pressure liquid chromatography.

Abstract Nucleotide profiles of cell extracts were determined using high pressure liquid chromatography. The nucleotide levels were quantitatively reproducible and the adenine levels as determined by the analyzer were in close agreement with those determined by an enzymatic cycling procedure. Optimal conditions were determined for the mono-, di- and triphosphates of the naturally occurring ribosides as well as for the sulfur analogs of some of these compounds. The peaks were identified by the use of internal standards, by comparison to chromatograms of standard solutions, by collection and identification of the fractions chemically and spectrophotometrically and by the use of an enzymic peak-shift technique. The latter method which utilizes the specificity of enzyme reactions with a nucleotide or class of nucleotides, can be used not only to verify peak identities but also to clarify or “unmask” a chromatogram. Chromatograms of cell extracts of red blood cells, homogenized schistosomes or murine leukemia or sarcoma cells were obtained in seventy minutes.

Purine metabolism in primitive erythrocytes

Abstract 1. 1. The activities of AMP kinase, GMP kinase, NDP kinase, PNPase and HGPRTase were determined in the erythrocytes of seal, eel, hagfish and dogfish. 2. 2. Erythrocytic NDP kinase activities were high and electrophoretically heterogeneous in all species examined. 3. 3. Patterns of nucleotide extracts of whole blood and erythrocytes in seal, sand dab, skate, hagfish, dogfish, nurse shark and stingray were similar when determined by high-pressure liquid chromatography. Patterns of the whole blood of eel and lemon shark, however, were strikingly different in containing large amounts of GDP and GTP.

Rates of RNA synthesis during early embryogenesis in Drosophila melanogaster

Abstract We determined the absolute rates of RNA synthesis during embryogenesis in Drosophila melanogaster by measuring the incorporation of 3 H-5-orotic acid into RNA, and the specific activity of the UTP pool. Initially (preblastoderm) the rate of RNA synthesis is relatively high, but declines to a lower level by gastrulation. The data suggest that RNA synthesis is initiated during very early embryogenesis.

Pathways of nucleotide metabolism in schistosoma mansoni—IV: Incorporation of adenosine analogs in vitro☆

Abstract The incorporation in vitro of adenine or adenosine analogs into schistosome nucleotides is demonstrated. Tubercidin, 2-fluoroadenosine and 2-fluoroadenine were all shown to be converted into analog triphosphate nucleotides. Since tubercidin and 2-fluoroadenosine are not substrates for adenosine deaminase or purine nucleoside phosphorylase and are not susceptible to degradation to the free base level, it is assumed that they are converted to nucleotides by reaction with adenosine kinase. The incorporation of 2-fluoroadenine into the nucleotide pools indicates that it serves as a substrate for adenine phosphoribosyltransferase. Tubercidin, added to the culture medium, interferes with the maintenance of normal ATP levels. When the concentration of the analog greatly exceeded that of adenine or adenosine in the medium, virtual shutdown of adenosine triphosphate synthesis followed. It is suggested that stoichiometric competition for enzyme sites may determine the relative amounts of nucleotides formed.

The reactions of 1, 3-dimercaptopropane, lipoic acid, and dihydrolipoic acid with metal ions

Abstract Solid compounds of α-lipoic acid with Hg2+, and of dihydrolipoic acid and 1,3-dimercaptopropane with Ni2+ and Hg2+ have been prepared. For comparative purposes, qualitative studies have been carried out on the reactions of other metal ions (Zn2+, Cu2+, Co2+ and Pb2+) with these sulfur compounds. With lipoic acid, the product formed readily with Hg2+, but photolytic or thermal cleavage of the disulfide link was necessary before any isolatable products were formed with the other cations. With dimercaptopropane and dihydrolipoic acid, products were formed with all the cations. The importance of these systems is related to the use of lipoic acid in the treatment of heavy metal poisoning.

Nucleotide metabolism in the whole blood of various vertebrates: enzyme levels and the use of high pressure liquid chromatography for the determination of nucleotide patterns

Abstract 1. 1. The nucleotide profiles of whole blood of man and animals were obtained by high pressure liquid chromatography. 2. 2. Within a species, the chromatograms were remarkably reproducible; however, each species exhibited a variation in the pattern that was characteristically its own. 3. 3. The major difference in higher and lower species was in the distinguishable 4. amounts of GTP and UTP in the lower species and the virtual absence of these compounds in the higher species. 5. 4. Adenylate kinase, guanlate kinase, nucleoside diphosphokinase and purine nucleoside phosphorylase activity were determined in the erythrocytes of some of these species.

DNA synthesis during early development of Drosophila melanogaster

Abstract The synthesis of DNA in D. melanogaster embryos was followed from preblastoderm to gastrula stages using in vitro radiolabelling techniques. Labelled thymidine was incorporated into main-peak nuclear DNA and into a satellite DNA species with a buoyant density of 1·689 g· cm−3 in CsCl. Mitochondrial DNA was not significantly labelled in pre- or postgastrula embryos. Analyses of changes in size and specific activity of the deoxythymidine triphosphate pool in developing embryos were made by high-pressure liquid chromatography and used for the interpretation of temporal changes in patterns of uptake of labelled thymidine into total and acid-insoluble radioactivity.

Incorporation of purine analogs into the nucleotide pools of human erythrocytes.

For the past decade this laboratory has devoted much of its attention to an examination of various facets of purine metabolism in human erythrocytes. These cells do not have the complete pathway for the de novo synthesis of purines and do not make nucleic acids. On the other hand, they have an active nucleotide metabolism and contain the salvage enzymes, hypoxanthine-guanine phosphoribosyl transferase (HGPRTase), adenine phosphoribosyl transferase (APRTase) and adenosine kinase. In view of the fact that the activities of certain enzymes of purine metabolism are quite high (e.g., purine nucleoside phosphorylase occurs at a level of about 15 umolar units/ml of erythrocytes) and the total mass of erythrocytes in the adult human being is in excess of two liters, it appears that these cells play an important and perhaps not yet fully appreciated role in the whole body economy of purines in man. Therefore, we believe that the human erythrocyte provides a very useful model system for the examination of purine metabolism in man as well as for investigations of the action of certain purine and purine nucleoside antimetabolites, many of which are important in medicine.

The Use of High Pressure Liquid Chromatography to Monitor Nucleotide Levels in Cells

High pressure liquid chromatography has proved to be a valuable tool in measuring the nucleotide levels in cell extracts (1, 2). The analyses are accomplished with high speed, sensitivity, resolution and accuracy. A decided advantage of this technique is that the major naturally occurring nucleotides can be monitored at one time concurrently with nucleotides of purine and pyrimidine analogs.