Pyrophosphate, inorganic, labeled

A mechanism proposed 87) for the alkaline hydrolysis of tetraethyl pyrophosphate, which is markedly accelerated by HPO e ions, has been substantiated by isotopic labeling 88). The nucleophilic attack by HPOJp on the symmetrical pyrophosphate 131 is considered to lead initially to the unsymmetrical P P1-diethyl pyrophosphate dianion 132 which decomposes spontaneously under the conditions of reaction to give the diethyl phosphate anion and POf 102. The latter reacts with water to form inorganic phosphate and with alcohols suclj as methanol and ethylene glycol to produce alkyl phosphates. 

Bicarbonate must be activated by the enzyme toward carboxyl transfer. The requirement for ATP and the transfer of labelled oxygen from bicarbonate to inorganic pyrophosphate indicate activation by phosphorylation (Ochoa et ai, 1962). In addition, the most nucleophilic site of biotin is the ureido oxygen. The ureido nitrogen of biotin is a poor nucleophile, and any proposed enzymic mechanism must account for enhancement of the nucleo-philicity of N(T) relative to 0(2 ). 

The lipid-soluble intermediate that accumulates is dolichol pyrophosphate, whose terminal phosphate comes from glucose 1-phosphate. (See Figure 11.23 in Section 11.3.3 in the text.) Bacitracin is an antibiotic that forms a 1 1 complex with dolichol pyrophosphate, preventing its hydrolysis to dolichol phosphate and inorganic phosphate. Thus, in the presence of bacitracin, the label will remain in dolichol pyrophosphate. In the absence of bacitracin, the terminal phosphate will be released as inorganic phosphate. 

Labeling of DNA with P by nick translation. The diagram shows the replacement of a single nucleotide residue. In the nick translation procedure many such replacements occur throughout the DNA molecule. dR 2 -deoxyribose. PP Inorganic pyrophosphate. 

Synthesis of DNA. An enzymatic synthesis of polymers of deoxy-ribonucleotides has been described recently. Enzyme preparations from E. coli form acid-insoluble products that are digestible by DNAase. Study of the reaction was complicated by multiple requirements, but extensive purification has permitted the determination of some of the properties of the system. The enzyme is relatively inactive when a single nucleotide is used as a substrate, and appears to have a requirement for the simultaneous presence of four deoxynucleoside triphosphates, deoxy ATP, deoxy GTP, deoxy CTP, and deoxy TTP. Only the triphosphates, not the corresponding disphosphates, are active. In addition to nucleotides, a primer is required. DNA from various sources serves as primer Mg++ is also required. The products of the reaction are polymer and inorganic pyrophosphate. Reversibility has been indicated by experiments in which labeled inorganic pyrophosphate was incorporated into the nucleotides. 

The enzyme has been called DPN pyrophosphorylase, mnce it has been shown that incubation of DPN with radioactive inorganic pyrophosphate leads to the synthesis of ATP with only the two terminal phosphates labeled... 

In liver and red corpuscles (see page 124), the rate of rejuvenation of the labile adenylpyrophosphate phosphorus is of about the same order of magnitude as that in resting muscles. Five minutes after intravenous injection of labeled phosphate, the specific activity of the pyrophosphate phosphorus amounts to 83% of that for the inorganic phosphorus, and a similar figure was found for pyrophosphate present in red corpuscles of the rabbit (87). 

Fig. 10. Two-dimensional correlated spectrum for a mixture of phosphate metabolites in separate pH compartments. The experiment employed concentric NMR tubes with the inside (capital letters) at pH 7.5 and the outside (lower-case letters) at pH 6.5. The diagonal peaks labeled D-d, E-e, F-f, and G-g, represent the phosphate resonances of )S-ATPjJ, AMP, inorganic phosphate, and pyrophosphate, respectively, (iross-peak A-a correlates the a- and / phosphorus resonances of ADP, B-b correlates the y and P resonances of ATP, and C-c correlates the a and p resonances of ATP. The resonances were broadened by the addition GdOj. From Van Divender and Hutton (1982).

Fig. 11. Two-dimensional correlated spectrum of the following mixture ATP, ADP, AMP, inorganic phosphate, pyrophosphate, adenosine tetraphosphate (A4P), diadenosine 5, S"-tetraphosphate (AP4A), and GdCl,. The labeling is consistent with Fig. 10 with the following additions diagonal peaks k and 1 (—23.7 and —23.9 ppm) represent the P- and y-phosphorus resonances of A4P and the p resonances of AP4A, respectively the off- diagonal peak h correlates the

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