Purification of protoplast-secreted acid phosphatase from baker's yeast Effect on adenosine triphosphatase activity

Mildner, P.; Barbarić, Slobodan; Z, Golubić; Ries, Blanka

doi:10.1016/0005-2744(76)90050-4

Cited by 16 publications

(3 citation statements)

References 8 publications

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“…This was confirmed by periodic acid-Schiff staining of IAP's αand β-subunits ( Figure 2D), and provides a rationale for the anomalous molecular mass (50 kDa) estimated for IAP during FPLC of the native enzyme on a calibrated Superose 12 column. The unusual behaviour of potato tuber and yeast APs during gel filtration chromatography has been attributed to glycosylation [27,31].…”

Section: Discussionmentioning

confidence: 99%

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

Bozzo

Raghothama

Plaxton

2004

Biochemical Journal

138

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An intracellular acid phosphatase (IAP) from P(i)-starved (-P(i)) tomato ( Lycopersicon esculentum ) suspension cells has been purified to homogeneity. IAP is a purple acid phosphatase (PAP), as the purified protein was violet in colour (lambda(max)=546 nm) and was insensitive to L-tartrate. PAGE, periodic acid-Schiff staining and peptide mapping demonstrated that the enzyme exists as a 142 kDa heterodimer composed of an equivalent ratio of glycosylated and structurally dissimilar 63 (alpha-subunit) and 57 kDa (beta-subunit) polypeptides. However, the nine N-terminal amino acids of the alpha- and beta-subunits were identical, exhibiting similarity to the deduced N-terminal portions of several putative plant PAPs. Quantification of immunoblots probed with rabbit anti-(tomato acid phosphatase) immune serum revealed that the 4-fold increase in IAP activity due to P(i)-deprivation was correlated with similar increases in the amount of antigenic IAP alpha- and beta-subunits. IAP displayed optimal activity at pH 5.1, was activated 150% by 10 mM Mg(2+), but was potently inhibited by Zn(2+), Cu(2+), Fe(3+), molybdate, vanadate, fluoride and P(i). Although IAP demonstrated broad substrate selectivity, its specificity constant ( V (max)/ K (m)) with phosphoenolpyruvate was >250% greater than that obtained with any other substrate. IAP exhibited significant peroxidase activity, which was optimal at pH 9.0 and insensitive to Mg(2+) or molybdate. This IAP is proposed to scavenge P(i) from intracellular phosphate esters in -P(i) tomato. A possible secondary IAP role in the metabolism of reactive oxygen species is discussed. IAP properties are compared with those of two extracellular PAP isoenzymes that are secreted into the medium of -P(i) tomato cells [Bozzo, Raghothama and Plaxton (2002) Eur. J. Biochem. 269, 6278-6286].

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Section: Discussionmentioning

confidence: 99%

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

Bozzo

Raghothama

Plaxton

2004

Biochemical Journal

138

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“…As the Pho5 enzyme is located in the periplasm between the plasma membrane and cell wall in soluble form, its activity can be easily monitored with whole cells (37–39). It is non-essential and mutations do not matter under standard laboratory growth conditions making it amenable to extensive mutational analysis.…”

Section: Classical Studies: Past and Presentmentioning

confidence: 99%

The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

Korber

Barbarić

2014

Nucleic Acids Research

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Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks.

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“…Structural genes for these enzymes are PHO8, PHO5, and PHO3, respectively (2, 5, 6). The repressible acid phosphatase is an exocellular, multimeric glycoprotein with an average mass of 490 kilodaltons (7,8), consisting of several sequence-related polypeptides. These variant polypeptides, probably forming different isoenzymes in a heterologous quaternary structure, are encoded by three distinct mRNAs (9), distinguishable by their antigenic products in cell-free translation (p60, p58, and p56).…”

mentioning

confidence: 99%

Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.

Rogers¹,

Lemire²,

Bostian³

1982

Proc. Natl. Acad. Sci. U.S.A.

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Two clones from a A phage collection containing yeast genes regulated by inorganic phosphate were shown by lowstringency hybridization to select three mRNAs that direct the in vitro synthesis of repressible acid phosphatase (EC 3.1.3.2) polypeptides p60, p58, and p56. By higher stringency hybridization one yeast fragment [8 kilobases (kb)] selects p60 mRNA and the other (5 kb) selects p56 mRNA. These EcoRI digestion fragments were subcloned in yeast transformation vectors and hybridization selection assignments were confirmed by measuring enzyme and mRNA levels in transformants. Enzyme and mRNA levels in (8-kb) high copy number transformants grown in high inorganic phosphate medium revealed a hitherto undetected acid phosphatase protein, P57, which is believed to correspond to the constitutive enzyme encoded by PHO3. The identity of the 8-kb fragment purported to contain the PHO5/PH03 genes was confirmed by genetic mapping of an integrated copy of this fragment. The site of integration of the 5-kb fragment was demonstrated to be unlinked to the PHO5/PH03 genes.In Saccharomyces cerevisiae a complex genetic system controls the expression of a number of phosphohydrolases in response to cellular levels of inorganic phosphate (P) (1)(2)(3)(4). Included in these enzymes is a cytoplasmic repressible alkaline phosphatase (EC 3.1.3.1), a repressible acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2], and an acid phosphatase synthesized during high-Pi growth. Structural genes for these enzymes are PHO8, PHO5, and PHO3, respectively (2, 5, 6). The repressible acid phosphatase is an exocellular, multimeric glycoprotein with an average mass of 490 kilodaltons (7,8), consisting of several sequence-related polypeptides. These variant polypeptides, probably forming different isoenzymes in a heterologous quaternary structure, are encoded by three distinct mRNAs (9), distinguishable by their antigenic products in cell-free translation (p60, p58, and p56). We previously established that expression of these mRNAs is regulated by Pi and that repressible APase is synthesized de novo upon derepression (9), consistent with a transcriptional regulation model proposed by Oshima and colleagues (10). According to their genetic model, PH05 and PH08 are transcriptionally regulated by a coordinate mechanism involving the products of numerous regulatory genes that interact at the posttranslational level. PH03 expression is mediated by two genes independent of this mechanism.Recently, several yeast genes under Pi control were isolated by Kramer and Andersen (11). Two of these genes, defined by EcoRI restriction endonuclease fragments of 8 and 5 kilobases (kb), are regulated in a fashion characteristic of phosphatase structural genes. We have established the identity and chromosome location of these transcriptionally regulated genes, using yeast transformation and hybridization selection. We demonstrate here that the 8-kb EcoRI fragment contains two phosphatase structural genes, one encoding ...

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Purification of protoplast-secreted acid phosphatase from baker's yeast Effect on adenosine triphosphatase activity

Cited by 16 publications

References 8 publications

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

Structural and kinetic properties of a novel purple acid phosphatase from phosphate-starved tomato (Lycopersicon esculentum) cell cultures

The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.

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