Nucleotides acid-labile phosphate

The methods for measuring these nucleotides are based on two distinctive properties of these cofactors. These are (1) their characteristic absorption at 260 m/i and (2) the fact that ADP and ATP have one and two acid-labile phosphate groups, respectively. For example, the pyrophosphate linkages of ATP are completely hydrolyzed to orthophosphate in 10 min. at 100° (IN acid), whereas the hexose-6-phosphates are not appreciably hydrolyzed by this treatment. 

Membrane preparations from the alga Prototheca zopfii incorporate D-mannose and 2-acetamido-2-deoxy-D-glucose from their respective sugar nucleotides into acid labile glycolipids whose properties are consistent with those of dolichyl D-mannopyranosyl phosphate. The intermediate appears to be involved in the formation of glycoproteins containing high proportions of D-mannosyl residues of the type present in more-evolved eukaryotes. 

Citrullinase. The bacterial enzyme that degrades citrulline has been called citrullinase, citrulline ureidase and citrulline phosphorylase. The reaction requires inorganic phosphate, Mg++, and ADP, and ATP is formed together with NH3, CO2, and ornithine. Arsenate supports the breakdown of citrulline in the absence of phosphate and adenine nucleotides. The mechanism of the phosphorolysis was shown by Jones et to be straightforward the first products are ornithine and carbamyl phosphate, which had previously been considered to be too unstable to exist free. Carbamyl phosphate transfers its phosphate to ADP in a reversible kinase reaction, and the carbamyl group also can react with ornithine to form citrulline (VI). These reversible reactions explain the requirement for stoichiometric amounts of adenine nucleotides for a coupled reaction to remove the labile phosphate. Carbamic acid may equilibrate non-... 

The lability of the 2 - and 3 - nucleotides to acids may be compared to the acid lability of glycerol 1-phosphate the production of a mixture by the alkaline hydrolysis of nucleic acids is similar to the alkaline hydrolysis of glycerol monomethyl 1-phosphate to methanol and glycerol 1- and 2-phosphates 122),... 

Step 3. Alkali dissolves most of the RNA-DNA-protein residue and hydrolyzes RNA quantitatively to mononucleotides (91). All four of these contain phosphorus but only the purine nucleotides react in the conventional ribose assay. Alkali also releases phosphoprotein phosphorus as inorganic phosphate (91) (the degree of completeness has not been reported). DNA is alkali resistant as long as the purine-desoxyribose linkages are intact (these bonds are acid labile—see comments under step 1). Once adenine and guanine are removed, alkali lability of DNA appears, due presumably to a cyclization mechanism similar to that which is responsible for RNA lability (14). 

The isomerism existing between the pairs of nucleotides was attributed to the different locations of the phosphoryl residues in the carbohydrate part of the parent nucleoside,49 63 since, for instance, the isomeric adenylic acids are both hydrolyzed by acids to adenine, and by alkalis or kidney phosphatase to adenosine. Neither is identical with adenosine 5-phosphate since they are not deaminated by adenylic-acid deaminase,68 60 and are both more labile to acids than is muscle adenylic acid. An alternative explanation of the isomerism was put forward by Doherty.61 He was able, by a process of transglycosidation, to convert adenylic acids a" and 6 to benzyl D-riboside phosphates which were then hydrogenated to optically inactive ribitol phosphates. He concluded from this that both isomers are 3-phosphates and that the isomerism is due to different configurations at the anomeric position. This evidence is, however, open to the same criticism detailed above in connection with the work of Levene and coworkers. Further work has amply justified the original conclusion regarding the nature of the isomerism, since it has been found that, in all four cases, a and 6 isomers give rise to the same nucleoside on enzymic hydrolysis.62 62 63 It was therefore evident that the isomeric nucleotides are 2- and 3-phosphates, since they are demonstrably different from the known 5-phosphates. The decision as to which of the pair is the 2- and which the 3-phosphate proved to be a difficult one. The problem is complicated by the fact that the a and b" nucleotides are readily interconvertible.64,64... 

It remains to be explained why treatment of the protected nucleotide (153) with ammonium hydroxide produces cytidine 2 -phosphate only, whereas similar treatment of (156) gives a mixture of the 2 - and 3 -isomers. A possible explanation may reside in differences in the lability of the benzoyl group on 0-2 or 0-3 of ribonucleosides, the 2 -benzoate being the more labile to ammonium hydroxide. Such a hypothesis is in accordance with the observation that partial debenzoylation of iV -benzoyl-tri-0-benzoylcytidine yields a mixture containing (152), but not (155). It had been noted that, in ribonucleosides, the 2 -hydroxyl group is more acidic than the 3 - and 5 -hydroxyl groups. It would be expected that 2 -0-acyl derivatives of ribonucleosides should be relatively more susceptible to alkaline hydrolysis than the 3 - or 5 -0-acyl derivatives. 

Even though orotidylic acid or orotidine was implicated in pyrimidine formation, the precise role of orotic acid per se remained to be evaluated. On the other hand, it was posable that orotic acid was a normal intermediate that condensed with a ribose compound to yield orotidine or orotidylic acid during the biosynthetic process. In support of this thesis, it was found that 5-phosphoribosyl-l-pyrophosphate was utilized for nucleotide formation from orotic acid (83). On the other hand, it was equally posable that an aliphatic compound, such as aminofumaric acid (335) or carbamylaspartic acid (339), could have coupled with a ribose compound and formed orotidine or orotidylic acid directly without the existence or participation of orotic acid per se. In this latter instance, orotic acid would not be conadered a true intermediate in pyrimidine biosynthesis but merely an accidental cleavage product of hi ly labile orotidine or orotidylic acid. At this time research in the area of purine biosynthesis indicated that a series of acyclic intermediates attached to ribose 6-phosphate were biosynthetic intermediates and that free purines per se were not (Section II, B.). 

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