AMPS purification

Biomedical Applications. TRIS AMINO is used for a number of purposes in its pure form, it is an acidimetric standard the USP grade can be utilized intraveneously for therapeutic control of blood acidosis TRIS AMINO also is useful in genetic engineering as a buffering agent for enzyme systems, industrial protein purification, and electrophoresis. AMP has found use as a reagent in enzyme-linked immunoassays. The primary appHcation is for alkaline phosphatase assays. 

EC 1.1.1.27]. 40-Fold purification by affinity chromatography using Sepharose 4B coupled to 8-(6-aminohexyl)amino-5 -AMP or -NAD. [Lees et al. Arch Biochem Biophys 163 561 7974 Pesce et al. J Biol Chem 239 1753 7964.]... 

Protamine kinase (from rainbow trout testes) [37278-10-7] [EC 2.7.1.70]. Partial purification by hydoxylapatite chromatography followed by biospecific chromatography on nucleotide coupled Sepharose 4B (the nucleotide was 8-(6-aminohexyl)amine coupled cyclic-AMP). [Jergil et al. Biochem J139 441 1974.]... 

Because many cells maintain ATP, ADP, and AMP concentrations at or near the mass action ratio of the adenylate kinase reaction, the cellular content of this enzyme is often quite high. A consequence of such abundance is that, even after extensive purification, many proteins and enzymes contain traces of adenylate kinase activity. The presence of this kinase can confound the quantitative analysis of processes that either require ADP or are carried out in the presence of both ATP and AMP. Furthermore, the equilibrium of any reaction producing ADP may be altered if adenylate kinase activity is present. To minimize the effect of adenylate kinase, one can utilize the bisubstrate geometrical analogues Ap4A and ApsA to occupy simultaneously both substrate binding pockets of this kinase . Typical inhibitory concentrations are 0.4 and 0.2 mM, respectively. Of course, as is the case for the use of any inhibitor, one must always determine whether Ap4A or ApsA has a direct effect on a particular reaction under examination. For example. Powers et al studied the effect of a series of o ,co-di-(adenosine 5 )-polyphosphates (e.g., ApnA, where n =... 

The ligands used for enzyme purification can be specific to the desired enzyme (substrate, substrate analogue, enzyme inhibitor, antibody), specific for different classes of enzyme (AMP, NAD, PLP) or of limited predefined specificity (dye affinity chromatography withProcion, Cibacron dyes). 

Higashi, K. Fukunaga, K. Matsui, K. Maeyama, M. Miyamoto, E. Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase. Biochim. Biophys. Acta, 747, 232-240 (1983)... 

Dinbergs, I.D. Lindmark, D.G. Tritrichomonas foetus purification and characterization of hydrogenosomal ATP AMP phosphotransferase (adenylate kinase). Exp. Parasitol., 69, 150-156 (1989)... 

Batra, P.P. Burnette, B. Takeda, K. Purification and characterization of ATP AMP phosphotransferase from Mycobacterium marinum. Biochim. Biophys. Acta, 869, 350-357 (1986)... 

Ramotar, K. Pickard, M.A. AMP metabolism by the marine bacterium Vibrio (Benecka) natriegens purification and properties of adenylate kinase. Can. J. Microbiol., 27, 1053-1059 (1981)... 

The enzyme from the red marine alga seaweed, purified 950-fold catalyzes the deamination of compounds noted in Table VII 2 -AMP, 3 -AMP, NADP, and adenine were not substrates. Although no evidence regarding homogeneity was presented the constant ratios of activity for AMP ADP ATP NAD adenosine throughout the purification and heat inactivation data are consistent with a single enzyme. The reaction was activated by divalent cations Ca2+, Mg2+, and Ba2+ Ca2+ was twice as effective as Mg2+ and Ba2+. The percent activation by Ca2 of NAD, ATP, ADP, 5 -AMP, and adenosine deamination was 81, 260, 200, 116, and 0, respectively (182). 

This method combines the advantages of liquid chromatography with the selective and sensitive receptor assay Charm H-Q test. Milk sample previously precipitated (with Mcllvaine buffer) and preconcentrated on a C8 cartridge was fractionated using an LC system. The fractions were collected according to the retention times and peak widths, and they were assayed directly with Charm II-Q test with small modifications. This method was suited for the AMO, AMP, PenG, CLO, CEF, and CEP residues in the milk samples and after small modifications for CFD, TIC, and NAF. A simple purification scheme gave recoveries from 50% for AMO to 80-90% for other /3-lactams (95). 

Quantititatively, the value of an affinity chromatography step in the purification process is assessed by the purification factor and the recovery or yield of activity. The purification factor is the ratio of the specific activity after the affinity step to that before it, whereas the recovery or activity is the percentage of the initial activity in the sample that is recovered.61 Examples of the use of adenosine monophosphate (AMP) and related... 

Le Floc h, F. and Lafleuriel, J., The AMP aminohydrolase of Jerusalem artichoke tubers partial purification and properties, Physiol. Veg., 21, 15-27, 1983a. 

Cyclic AMP phosphodiesterase was partially purified from rat brain and from rat heart. The assay was applicable at different levels of enzyme purification. 

Cabezas A, Pinto RM, Fraiz F Canales J, Gonzalez-Santiago S, and Cameselle JC (2001) Purification, characterization, and substrate and inhibitor structure-activity studies of rat liver FAD-AMP lyase (cyclizing) preference for FAD and specificity for splitting ribonucleoside diphosphate-X into ribonucleotide and a five-atom cyclic... 

The insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

Precipitation with Ba(OH)2 and ZnS04 should not be carried out in the presence of high concentrations of ATP, as formation of cyclic AMP by a chemical reaction can occur [3]. Barium sulphate cannot be used for the purification of cyclic GMP solutions because it removes the nucleotide. 

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