Eduardo Recondo

Adenosine diphosphate glucose and starch synthesis

Received Septenber 19, 1961 In previous papers (Fekete et al., 1960; Leloir et al., 1961) an enzyme was described which catalyzes glucose transfer from uridine diphoaphate glucose (UD?G) to starch or oligosaccharides. Several synthetic nucleoside diphosphate sugars have now been tested with the same enzyme preparation. They were prepared following the procedures developed by Khorana and others (Roseman, Distler, Moffatt and Khorana, 1961) with alight modifications. The most interesting result was that adenosine diphosphate glucose (ADPG) reacts about tenfold faster than UDPG. A typical experiment is shown in figure 1.

SEPARATION OF SUGAR NUCLEOTIDES, PHOSPHORIC ESTERS AND FREE SUGARS BY PAPER CHROMATOGRAPHY WITH SOLVENTS CONTAINING BORATES OF ORGANIC BASES.

Abstract Four different chromatographic solvents containing borates of organic bases are described. Two of them permit the separation of sugar nucleotides differing only in the sugar moiety and give also a good resolution for some sugar 1-phosphates. The other two systems permit, in a single run, the separation of a group of sugars which is usually difficult to achieve.

Adenosine diphosphate glucose: Orthophosphate adenylyltransferase in wheat germ

Abstract An enzyme has been isolated from wheat germ which catalyzes the reaction: ADP-sugar + inorganic phosphate → ADP + sugar phosphate. Maximal activity was found at pH 8–9. The equilibrium of the reaction seems to be completely displaced towards ADP formation. The enzyme acts on ADP-glucose and deADP-glucose and more slowly, on ADP-xylose and ADP-β-glucose. Evidence is presented indicating that inorganic phosphate is incorporated in the terminal position of the nucleotide. Arsenate can be substituted for inorganic phosphate, AMP being the final product.

Enzymid dephosphorylation of adenosine diphosphate phosphoglyceric acid

Abstract An enzyme from muscle which acts on adenosine diphosphate phosphoglyceric acid and leads to the hydrolysis of the phosphate group at the 2-position of the glyceric acid moiety has been studied. The enzyme is strongly stimulated by inorgamoc pyrophosphate. This action is very specific. The enzyme also acts on 2,3-diphosphoglycerate.

Nucleotides in embryos in the stages of morula, gastrula, and neurula☆

Levels of nucleotides and sugar nucleotides in embryos of Bufo arenarum at the stages of morula, gastrula, and neurula have been measured. The total amounts of purine nucleoside diphosphates decreased from morula to gastrula, but increased sharply from gastrula to neurula. The levels of ADP followed this pattern, but those of GDP did not change significantly through the three stages. Purine nucleoside triphosphate levels, which had increased immediately after fertilization, remained almost constant through morula, gastrula, and neurula. As with the purine nucleoside diphosphates, the adenine nucleotide decreased from morula to gastrula, and increased from gastrula to neurula. In contrast, the level of GTP showed a sharp maximum at gastrula. The total pyrimidine nucleoside triphosphate did not change significantly from morula through neurula. As in previous stages of development, only uridine sugar nucleotides were detected. A sharp increase of the galactosyl ester of nucleotides was found at gastrula.

Antithrombin and first complement protein recognize the same active heparin fraction

Antithrombin (AT) high affinity of unfractionated heparin (UFH) resides in a specific pentasaccharide sequence. Heparin also regulates complement activity on the classical and the alternative pathways. Most experimental pieces of evidence accumulated show that these important activities reside in different segments of the heparin molecule. We demonstrated in previous papers that a low ionic strength and the presence of calcium ions are essential to detect specific interactions between glycosaminoglycans and proteins. Then these very strict conditions were used, and we demonstrated that the first protein complex of the human complement cascade recognizes in the UFH a fraction with very high anticoagulant activity. After isolation from the precipitate of the interaction, this fraction of heparin also contained the pentasaccharide sequence responsible for the great affinity with AT: in fact, it was strongly bound to a resin of AT agarose, and to detach it, an ionic strength of 0.6 M sodium chloride was required. In this way, the heparin regions responsible for the anticoagulant activity and also for the effects over the complement system were identified on the same short segment of the heparin molecule, which includes the active fraction of the glycosaminoglycan. The differences with early results could be explained by our experimental conditions of low ionic strength and the presence of calcium ions used for the interaction of the protein and the glycosaminoglycan.

Heparin and concanavalin A interaction: isolation of fraction with higher anticoagulant activity

Heparin and Concanavalin A complexes were studied under different conditions. Interaction was measured by reading the turbidity at 420 nm. The influence of the pH, heparin, and salt concentration was measured. In the presence of salts pH was very critical, and above pH 5.4 the interaction was practically negligible. At pH 4.6 or 5.2 and the lowest salt concentration compatible with buffering, heparin fractions with different anticoagulant specific activities were detected in the precipitate and in the supernatant, after the interaction. In all cases a significative difference was observed in favor of the heparin isolated from the precipitate. Possibility of an artifact was eliminated by using adequate blanks and running the coagulation tests in the presence of an excess of Concanavalin A.

A modified uronic acid-indole reaction

Abstract Modification of the conditions of the reaction of carbohydrates with indole in acidic medium made this reaction specific for uronic acids. A preliminary heating at 100° for 30 min in 3 M hydrochloric acid eliminated the interference of DNA and other 2-deoxyribose derivatives. The reaction was positive for a concentration above 17 nmol/mL of final solution, and the Lambert and Beer law was satisfied up to 86 nmol/mL. The spectrum of the reaction is identical to that obtained with DNA, with a maximum at 492 nm and a shoulder at 465 nm. d -Galacturonic acid gave the highest value, while d -mannuronic acid gave 50%, and d -glucuronic acid only 14% of the value given by d -galacturonic acid. The reaction mechanism may be explained by dehydration of the uronic acid in 3 M hydrochloric acid at 100°, condensation of the dehydrated product with indole in the cold, and color formation after a second heating at 100°.

Nucleotides and nucleotide sugars in early development of bufo arenarum: Part I. mature oocytes

Abstract A study of nucleotides and nucleotide sugars in mature oocytes of Bufo arenarum has been performed. Two samples of oocytes from ovulations of ten females each were analyzed by ion-exchange chromatography and the following nucleotides and nucleotide sugars found 2 : NAD, UDPGNAc, UDPGalNAc, UDPG, UDP, NADP, CTP, UTP, UDPGA, ADP, GDP, GTP, and ATP. The nucleotides and sugar-nucleotides were identified on the basis of chemical, enzymic, and chromatographic analysis. To minimize the decomposition of unstable substances, the extraction was performed with trichloroacetic acid in the cold, the contact of the sample with the acid was minimal and ammonium chloride was used for the elution from the Dowex 1-resin. In order to detect compounds present in minute amounts, very large samples were used (60,000 to 120,000 oocytes). A nucleotide of complex structure, which binds weakly to the column, was detected in the two samples analyzed.

Nucleotides and nucleotide sugars in human blood cells. II. Group "O".

Abstract A study of nucleotides and nucleotide sugars in human blood cells has been carried out. Ten samples from healthy yound people of group “O” were analyzed by ion-exchange chromatography, and the following nucleotides and nucleotide sugars were found: NAD+; UDP-glucose, UDP-mannose, UDP-galactose, UDP-acetylglucosamine and UDP-acetylgalactosamine; AMP; NADP+; ADP-glucose, ADP-galactose and ADP-mannose; GDP-mannose and GDP-fucose; ADP and ATP. The sugar nucleotides were identified on the basis of chemical, enzymatic and chromatographic analysis. UDP-galactose, UDP-mannose, UDP-acetylgalactosamine, ADP-glucose, ADP-galactose, ADP-mannose, GDP-mannose and GDP-fucose have not been found before in human blood, and UDP-mannose was not known to occur in nature. In order to minimize the decomposition of unstable substances, mild techniques were applied: cold acid extraction and neutral (ethanol-chloroform) extraction were compared, and in both cases the aqueous phase was passed through a column of Dowex- i (Cl-form) resin, and the nucleotides eluted with a linear gradient of NH4Cl. Results showed that the extraction step is not very critical, but the use of ammonium salts and almost neutral pH, as compared with methods used in previous papers, permitted the isolation of ten nucleotide-linked sugars, eight of which have not been described before in human blood. An adenine nucleotide with very particular properties was extracted with the ethanol-chloroform technique; it was eluted by the concentration of NH4Cl necessary to elute ATP, but it behaved as AMP on paper chromatography in several solvent systems. It is very unstable and decomposes even at −20° in a few days.