Phosphorylases sources

An extreme example of the reduction in radiation damage is that of data collection at the SRS on purine nucleoside phosphorylase. On a conventional source usually a crystal can give only one 3 A resolution still photograph before the crystal suffers serious damage. At the synchrotron three crystals will give a comptete set of 4 equivalent reflections to a resolution limit of 3 A (see case study below)... 

Residues in the region of the substrate-binding site for phosphate in phosphorylase b in the T state (a) and the R state (b). The side chain of Asp 283 leaves the binding site in the R structure, and the side chain of Arg 569 becomes available to interact with the phosphate. Key side chains and bound phosphate anion are shown in red. Portions of the polypeptide backbone are drawn in heavy black. (Source From D. Barford and L. N. Johnson, The allosteric transition of glycogen phosphorylase, Nature 340 609, 1989.)... 

After purine nucleotides have been converted to the corresponding nucleosides by 5 -nucleotidases and by phosphatases, inosine and guanosine are readily cleaved to the nucleobase and ribose-1-phosphate by the widely distributed purine nucleoside phosphorylase. The corresponding deoxynucleosides yield deoxyribose- 1-phosphate and base with the phosphorylase from most sources. Adenosine and deoxyadenosine are not attacked by the phosphorylase of mammalian tissue, but much AMP is converted to IMP by an aminohydrolase (deaminase), which is very active in muscle and other tissues (fig. 23.20). An inherited deficiency of purine nucleoside phosphorylase is associated with a deficiency in the cellular type of immunity. 

Phosphorolysis of ribonucleic acid with polynucleotide phosphorylase gives a mixture of the diphosphates of the four common nucleosides, which are transformed into triphosphates with enolpyruvate phosphate and pyruvate kinase. This mixture may be used as such as a source of uridine triphosphate in the preparation of the nucleotide-sugar uridine 5 -(a-D-glucopy-ranosyl diphosphate) ( uridine-diphosphate-glucose, UDP-Glc), or as a... 

Position of Bond Cleavage PNP (EC 2.U.2.1) from human erythrocytes (homogeneous, purified by formycin B affinity chromatography) as well as from E, coli were allowed to equilibrate a mixture of R-l-[1 0lj]-P, pl Oij, hypoxanthine and inosine at pH 7-00 in 10 mm NMR tubes. The chemical shift differences of the 31p nuclei of the two R-l-P s (13 9 Hz for the human erythrocytic and 13.1 Hz for the E. coli enzyme) as well as of the two P3 resonances (13 9 Hz for erythrocytic and 13-7 Hz for E. coli source) clearly indicated C-0 bond cleavage by these enzymes as well. In addition, no evidence was found over the time course of the NMR measurements (l hr) for purine nucleoside phosphorylase catalyzed exchange of pl o + H2O (solvent) J randomized P. Therefore,... 

Earlier studies on the properties of phosphorylases isolated from various sources have indicated that their subunits are similar in size with about 100,000 daltons.15-17 The reaction proceeds in a rapid equilibrium random Bi-Bi mechanism as has been shown by kinetic studies with rabbit skeletal muscle phosphorylases a18-20 and b,21,22 rabbit liver enzyme,23 potato tuber enzyme,24 and the enzyme from E. coli.25) In contrast, the substrate specificities for various glucans differ considerably depending on the enzyme sources. The rabbit muscle enzyme has high affinity for branched glucans such as glycogen and amylopectin but low affinity for amylose and maltodextrin.26,27 The potato tuber enzyme can act on amylose, amylopectin, and maltodextrin but only poorly on glycogen,28,29 while the E. coli enzyme shows high affinity for maltodextrin.10 ... 

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. 

Evidence for the presence of hyperglycemic factors in the CC were reported first in P. americana (1) and supported subsequently in B. discoidalis (3). The HGHs act on the fat body, the synthetic source for trehalose in insects (22), to elevate phosphorylase activity and the conversion of glycogen stores to the precursors for trehalose synthesis (2,3). Initially, it was believed that HGHs activated phosphorylase via the synthesis of adenosine 3 5 -cyclic monophosphate (cAMP) in the same manner that glucagon or epinephrine activate liver phosphorylase in vertebrate animals (23). Injections of intact adult P. americana with synthetic Pea-CAH-I and -II result in a 50% net increase in fat body cAMP over water-injected controls accompanied by a more than 3-fold increase in fat body phosphorylase activity (24). However, the CAHs fail to elevate cAMP levels of fat bodies from P. americana in vitro even though both phosphorylase activity and trehalose synthesis increase (25). In the latter case, Ca + is essential for the action of the CAHs, and its omission from the incubation medium inhibits the hypertrehalosemic response. 

Phosphorylase is an enzyme of wide, if not universal, occurrence in plants. It is also found in animal skeletal muscle, liver, and heart. In animals, phosphorylases usually exist in two forms, the a form and the b form. The b form is converted enzymically into the active a form by phosphorylation. Phosphorylases isolated from different sources appear to differ in structure, but it is not yet known whether they differ in action pattern. 

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... 

Pyrimidines derived from dietary or endogenous sources are salvaged efficiently in mammalian systems. They are converted to nucleosides by nucleoside phosphorylases and then to nucleotides by appropriate kinases. 

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