Phosphorylases bacterial

TRETHEWEY, R.N., FERNIE, A.R., BACHMANN, A., FLEISCHER-NOTTER, H., GEIGENBERGER, P, WILLMITZER, L., Expression of a bacterial sucrose phosphorylase in potato tubers results in a glucose-independent induction of glycolysis, Plant Cell Environ., 2001, 24, 357-365. 

The dried bacteria also contain invertase and this hydrolytic enzyme competes with the sucrose phosphorylase for sucrose. However, it is possible to eliminate most of the invertase from the bacterial preparations by several precipitations with ammonium sulfate. Using a partially purified sucrose phosphorylase preparation and a mixture of... 

Covalent interconversion of enzymes is well established as a fundamental theme in metabolic regulation. The prototypic reversible interconverting systems include the sequence of phosphorylation/dephosphorylation steps in the activation of mammalian glycogen phosphorylase and pyruvate dehydrogenase as well as the nucleotidyla-tion/denucleotidylation using UTP and ATP in the bacterial glutamine synthetase cascade (see Fig. 1.). 

In 1955, Marianne Grunberg-Manago and Severo Ochoa discovered the bacterial enzyme polynucleotide phosphorylase, which in vitro catalyzes the reaction... 

Factors which tend to decrease bioavailability of pyridoxine include (1) Administration of isoniazid (2) loss in cooking (estimated at 30-45%)—vitamin is water-soluble, (3) diuresis and gastrointestinal diseases (4) irradiation. Availability can be increased by stimulating intestinal bacterial production (very small amount), and storage in liver. The target tissues of Be are nervous tissue, liver, lymph nodes, and muscle tissue. Storage is by muscle phosphorylase (skeletal muscle—small amount). It is estimated that 57% of the vitamin ingested per day is excreted. The vitamin exerts only limited toxicity for humans. 

The first enzyme discovered that could catalyze polynucleotide synthesis was a bacterial enzyme called polynucleotide phosphorylase. This enzyme, isolated by Severo Ochoa and Marianne Grunberg-Manago in 1955, could make long chains of 5 -3 -linked polyribonucleotides starting from nucleoside diphosphates. However, there was no template requirement for this synthesis, and the sequence was uncontrollable except in a crude way by adjusting the relative concentrations of different nucleotides in the starting materials. 

Fig. 6.4 Affinity electrophoresis of the recombinant phosphorylases from potato tuber.,3) A crude bacterial cell extract containing the type-L isozyme (lane 1), the chimeric enzyme (lane2), or the type-H isozyme (lane 3) was electrophoresed in 5% polyacrylamide gels supplemented with 0 (A), 50 (B), and 500 pg/ml (C) glycogen. After electrophoresis, the gels were stained for enzyme activity in KI-b solution. (Reproduced with permission from Goldsmith and Fletterick, Pure and Appl. Chern., 55(4), 583 (1983)).

In conclusion, it should be pointed out that in marked contrast to the very extensive studies on rabbit muscle phosphorylase, little attention has been paid to enzymes from other sources. However, primary structures of plant phosphorylases have now been determined and bacterial expression systems for the plant enzymes have also been made available as reviewed in this article. We hope that future studies on the structure and function of plant phosphorylases without allosteric regulation and comparison with those of the highly regulated animal enzyme will provide valuable information on this interesting group of enzymes, phosphorylases. 

Schinzel, R., and Nidetzky, N. 1999. Bacterial a-glucan phosphorylases. FEMS Microbiol. Lett., 171,73-79. 

Prepare a 500-ml filter flask to receive the potato extract by pouring 250 ml of F O into it and marking the position of the top of the fluid in the filtration reservoir with a felt-tipped pen. Pour out the F120. Suspend about 100 mg of phenylmercuric nitrate in a few milliliters of H20 and pour it into the filter flask. Phenylmercuric nitrate is added to inhibit other enzymes and to prevent bacterial growth during the incubation of potato phosphorylase with starch. 

The effect of temperature on the kinetics of the enzyme-catalyzed reaction is further complicated by changes in the pK s of the ionizable groups at the active center of the enzyme, thus producing additional changes in V and Km- A study of the variation of these pK s with temperature yields information on the heats of ionization of the ionizable group, AHj, by use of the equation AHj = —2.303 RT .dpK/ dT. The value of AHj has been calculated for the ionizing groups of bacterial alp/io-amylase and potato phosphorylase. ... 

Phosphorylases from animal, bacterial, and plant sources can be distinguished by their substrate preferences. Animal and bacterial phosphorylases prefer the short outer branches of highly branched cz-glucans, such as glycogen. The phosphorylase from Corynebacterium cal-luna, which accumulates a starch-like polysaccharide, is exceptional among bacterial enzymes... 

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