Acyl phosphates detection

The detection of minor amounts of monobenzyl phosphate, which presumably arise from hydrolysis of the monobenzyl acyl phosphate, is an example of the intramolecular migration of monoalkyl hydrogen phosphate. This process resembles the acid-catalyzed migration of a phosphoryl moiety adjacent to a vicinal hydroxyl function128-130, viz. 

Methanococcus voltae contains a membrane-bound vanadate-sensitive ATPase [48] that is inhibited by diethylstilbestrol, an inhibitor of eukaryotic P-type ATPases. The purified enzyme is composed of a single subunit (Mr 74 000), forms a covalent acyl-phosphate enzyme intermediate, and is not inhibited by nitrate or bafilomycin [49]. No such ATPase activity has been reported in other archaea. The presence of a second ATPase in M. voltae has been inferred since membranes react with antiserum prepared against the 3 subunit from the V-type ATPase of S. acidocaldarius [50]. Two peptides are detected whose Mr values (51 000 and 65 000) correspond to the masses for the two laigest subunits of the S. acidocaldarius ATPase [51]. There is evidence that ATP synthesis in the M. voltae enzyme is due to the operation of a sodium-translocating ATPase [50]. The relationship of the putative V-like ATPase to the sodium-translocating ATPase has not been established. 

A very clever three-phase test for the detection of metaphosphate intermediates in phosphoryl transfer reactions has been described by Rebek and coworkers (44). The basis of this test is the use of two polymers suspended in solution. The donor polymer contains a potential precursor to metaphosphate anion, e.g., an acyl phosphate or a phosphoramidate, and the recipient polymer contains an acceptor nucleophile, e.g., an amine. After reaction and physical separation of the polymers, the recipient polymer is analyzed for covalently bound phosphate. Since very few of the phosphoryl groups to be transferred will be on the surface of the donor polymer, detection of significant transfer to the recipient polymer provides evidence for a diffusible intermediate, i.e., free metaphosphate anion. Significant transfer did occur in dioxane or acetonitrile suspensions of the polymers, thereby providing evidence for an intermediate. However, this test for diffusible and, therefore, relatively stable metaphosphate anion is compromised by the choice of solvent. Both dioxane and acetonitrile can provide unshared electron pairs for the highly electrophilic metaphosphate anion such that the actual species that migrates from the donor polymer to the recipient polymer may be a complex between metaphosphate anion and the solvent. Such a role for solvent has been investigated stereochemically, the results of which will be described later in this section. 

The formation of hydroxamic acids is essentially irreversible and pulls the reaction to completion. ATP and similar phosphate compounds do not react with NH2OH. Reaction with hydroxylamine has long been used to determine acid derivatives such as esters, since hydroxamic acids form intensely colored complexes with ferric iron in acid solution. Acid anhydrides react much more readily than most other derivatives, and the hydroxamic reaction was introduced into biochemistry by Lipmann as a means for detecting acyl phosphate. Thioesters also react readily with... 

The anthocyanins exist in solution as various structural forms in equilibrium, depending on the pH and temperature. In order to obtain reproducible results in HPLC, it is essential to control the pH of the mobile phase and to work with thermostatically controlled columns. For the best resolution, anthocyanin equilibria have to be displaced toward their flavylium forms — peak tailing is thus minimized and peak sharpness improved. Flavylium cations are colored and can be selectively detected in the visible region at about 520 nm, avoiding the interference of other phenolics and flavonoids that may be present in the same extracts. Typically, the pH of elution should be lower than 2. A comparison of reversed-phase columns (Ci8, Ci2, and phenyl-bonded) for the separation of 20 wine anthocyanins, including mono-glucosides, diglucosides, and acylated derivatives was made by Berente et al. It was found that the best results were obtained with a C12 4 p,m column, with acetonitrile-phosphate buffer as mobile phase, at pH 1.6 and 50°C. 

Fig. 5. Biosynthesis of membrane phospholipids from alkyldihydroxyacetone-P (alkyl-DHAP), the first detectable intermediate formed in the biosynthetic pathway for ether-linked glycerolipids. Enzymes catalyzing the reactions are (1) NADPH alkyl-DHAP oxidoreductase, (II) acyl-CoA 1 -alkyl-2-lyso-sn-glycero-3-phosphate acyltransferase, (III) l-alkyl-2-acyl-in-glycero-3-phosphate phosphohydrolase, (IV) ATP 1-alkyl- /i-glycerol phosphotransferase, (V) CDP-choline l-alkyl-2-acyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), (VI) CDP-ethanolamine l-alkyl-2-acyI-sn-glycerol ethanolaminephosphotransferase, and (VII) acyl-CoA 1 -alkyl-2-acy 1-OT-glycerol acyltransferase.

Free fatty acids in human serum were derivatized with 1-naphthylamine after being converted to acyl chlorides [109], Serum (0.5 ml) was mixed with 0.1 ml of methanol containing an internal standard and 1.4 ml of 1/15 M phosphate buffer and poured into a column packed with Ig of Extrelut. The adsorbed fatty acid was recovered by elution with 10 ml of chloroform. After removal of solvent, the residue was dissolved in 0.6 ml of benzene. A solution of oxalyl chloride in benzene (2%) was added to the fatty acids and the mixture was allowed to react at 70 °C for 30 min. The solvent was removed at reduced pressure. Then naphthylamine solution (0.1ml) and triethylamine solution (0.01ml) were added. The reaction was carried out at 30 °C for 15 min, and 2 fi of the mixture was injected onto a pBondapak Cjg column at 40 °C. The mobile phase was methanol/water (81 19 v/v) and detection was at 280 nm Each fatty acid was quantitatively converted into its acid chloride and the overall recovery of naphthyl amides by this method was 94-106%. The main free fatty acids in human serum (14 0,16 0,16.1,18 0,18 1,18 2) and the internal standard (17 0) were separated in 30 min. 

At molecular level, the manifestations of the biological activity appear in specific biochemical reactions, conformational behavior, and dynamical properties of biomolecules. Experimental studies of various partially hydrated enzymatic proteins show that their activity accelerates rapidly at some critical hydration levels. Onset of the enzymatic activity of urease occurs at 0.15 g/g [469]. In the presence of chymotrypsin, the acylation reaction is undetectable at hydrations /i< 0.12 g/g, but its rate grows sharply above this critical hydration level [470]. The rate of enzymatic activity of glucose-6-phosphate dehydrogenase, hexoki-nase, and fumarase becomes detectable and start to increase sharply at hK 0.20 g/g, whereas this critical hydration is about 0.15 g/g for phosphoglucose isomerase [471]. Enzymatic activity of lysozyme can be detected only when hydration level achieves h 0.20 g/g [472, 473] (see Fig. 92). 

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