Yeast phosphatase

Alkaline phosphatase exists in two forms in animal tissues, one activated by added Mg(II) or Mn(II) and not inhibited by F or CN ions, the other independent of added divalent ions but inhibitable by cyanide . Yeast phosphatase, active at neutral pH, is activated by divalent metal ions in the order Mg > Mn > Ni, Co, Fe ". Metal analysis of purified kidney phosphatase reveals a mixture of bound ions Zn, Cu, Mn, Mg, and Fe . This variety of responses illustrates a classical problem in studies of enzyme activation by metal ions defining the species that is of greatest importance in vivo. 

Phosphatases capable of catalysing the dephosphorylation of TPP to thiamine have been encountered in animal tissues and in yeasts, and will probably be found in all living organisms. A kidney phosphatase and the acid yeast phosphatase (pH optimum ( . .4) have been shown to remove the phosphate groups one by one. The phosphatases cannot attack TPP when it is... 

The acid yeast phosphatase mentioned above is markedly inhibited by thiamine, and to a lesser degree by the pyrimidine part of the vitamin, not only when TPP is the substrate, but also when other phosphate esters, such as (X- or 8-glycerophosphate, are used. Animal phosphatases do not appear to be inhibited by thiamine o. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

The patient is started on fluconazole 400 mg/day, but 3 days later has persistent fever and develops hypotension and decreased urine output. Blood cultures reveal a germ tube-negative yeast growing in the blood. Laboratory studies revealed a white blood cell count of 12,300/mm3 (12x109/L), aspartate aminotransferase 68 IU/L (1.13 pKat/L), alanine aminotransferase 75 IU/L (1.25 pKat/L), alkaline phosphatase 168 IU/L (2.8 pKat/L), and normal bilirubin. Serum creatinine is 1.8 mg/dL (159 pmol/L). 

Figure 2. Regulators of the Cdc2 protein kinase. Schematic illustration of the regulators of Cdc2 kinase activity. Proteins that are circled indicate proteins that have been identified in higher eukaryotes only the squared proteins have been identified exclusively in the yeast systems. The remaining proteins have been identified in both higher and lower eukaryotes. Protein phosphatases are indicated by p tase.

Kinoshita, N., Yamano, H., Niwa, H Yoshida, T., and Yanagida, M. (1993). Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes Dev. 7 1059-1071. 

Kozulic, B., Barbaric, S., Ries, B., and Mildner, R (1984) Study of the carbohydrate part of yeast acid phosphatase. Biochem. Biophys. Rev. Comm. 122, 1083. 

Phosphoryl group transfer reactions add or remove phosphoryl groups to or from cellular metabolites and macromolecules, and play a major role in biochemistry. Phosphoryl transfer is the most common enzymatic function coded by the yeast genome and, in addition to its importance in intermediary metabolism (see Chapter 5), the reaction is catalysed by a large number of central regulatory enzymes that are often part of signalling cascades, such as protein kinases, protein phosphatases, ATPases and GTPases. 

Murray and Hunt, 1993). Cyclins, kinases, and phosphatases that regulate the passage of the cell through the G] — S phase transition are all present in mammals, invertebrates, and plants (Solomon, 1993 Doonan and Fobart, 1997 Zavitz and Zipursky, 1997). However, multicellular eukaryotes contain multiple orthologs of yeast cell cycle proteins they initiate proliferation via growth factors, rather than, for example, yeast mating factors, and they possess additional checkpoint controls and repair pathways. 

In yeast, H2A.X is dephosphorylated by the phosphatase PPH3p after it has been released from chromatin. The release of y-H2A.X from repaired chromatin is independent of DNA replication, and it therefore must be assumed that a chromatin remodeling complex actively exchanges 7-H2A.X from nucleosomes. Thus far, several candidate remodeling complexes have been identified that specifically target nucleosomes containing 7-H2A.X. 

Histone kinases responsible for N-phosphorylation have been isolated from regenerating rat liver [109] and Walker-256 carcinosarcoma cells [110]. One kinase with a pH optimum of 9.5 phosphorylated His-18 and His-75 of H4, while the other with a pH optimum of 6.5 phosphorylated lysine of HI. The enzyme from regenerating rat liver phosphorylated H4 at 1-phosphoryl histidine, while the carcinosarcoma enzyme phosphorylated H4 His at the position 3 [111]. Both kinases were cAMP independent [110]. Matthews and colleagues purified a 32-kDa histidine H4 kinase from yeast, Saccharomyces cerevisiae [112,113]. The enzyme phosphorylated His-75 (1-phosphoryl histidine) in H4. His-18 of H4 and other histidines in other core histones were not phosphorylated by this kinase [112]. Protein phosphatases 1, 2A, and 2C could dephosphorylate His-75 of H4 [114]. Applying a gel kinase approach to detect mammalian H4 histidine kinases, Besant and Attwood detected four activities in the 34-41 kDa range with extracts from porcine thymus [115]. 

Selected entries from Methods in Enzymology [vol, page(s)] Phosphatidate phosphatase from yeast, 197, 548 characterization and assay of phosphatidate phosphatase, 197, 553. 

It had been known from at least the time of Pasteur that the presence of sodium or potassium phosphate aided the progress of a yeast fermentation. Later intensive study showed that a complex group of enzymes (phosphatases and phosphorylases) was responsible for the phosphorylation, dephosphorylation and interconversion of D-glucose 6-phosphate, D-fructose 6-phosphate, D-fructose 1,6-diphosphate and similar substances in various types of cells and muscle tissue. Detailed reviews of the field are available. - A further advance was made in 1936, when Cori and Cori noted that in certain circumstances well-washed frog muscle immersed in a sodium phosphate buffer utilized the inorganic phosphate to produce a new hexose phosphate (the Cori ester). This compound was later shown to be a-D-glucopyranose-l-phosphate and yielded crystalline dipotassium and brucine salts. The Cori ester arose because... 

Fig. 1.36. Regulation of the sub-ceUular localization of the transcription factor SWI5 in yeast by phosphorylation. The subcellular localization of the SWI5 protein is regulated by phosphorylation/ dephosphorylation. In the phos-phorylated state, SWI5 is found in the cytoplasm, while in the under-phosphorylated state it is localized in the nucleus. Phosphorylation and dephosphorylation are catalyzed by either protein kinases or protein phosphatases and can be controlled via signal transduction chains.

Phosphorylation at Thrl4 and TyrlS leads to inactivation of the CDKs. In the fission yeast, the weel kinase is responsible for this phosphorylation and in mammals, there are enzymes homologous to wee-1 kinase. It is not clear whether this kinase performs both phosphorylations. Phosphorylation at Thrl4 and TyrlS is of particular importance for regulation of CDK activity in mitosis. The CDC2-cychn B complex is maintained in an inactive state imtil the end of G2 phase by the phosphorylation of Thrl4 and TyrlS. At the G2/M transition, the inactive state is ended by the action of CDC2S phosphatase, which cleaves off the inhibitory phosphate residues. 

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