Localization of the Cell-associated Phosphatase in Lysobacter enzymogenes

Tigerstrom, Richard G. Von; Stelmaschuk, Sheilah

doi:10.1099/00221287-131-7-1611

Cited by 10 publications

(9 citation statements)

References 22 publications

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“…In this case crude extracts are first subjected to an electrophoretic separation, and phosphatase activities are subsequently detected in situ by means of chromogenic reactions. Such reactions are based either on substrates which yield coloured products upon dephosphorylation [27,33,36,46] or on the detection of the released P i by means of the acidified ammonium molybdate method, which yields a blue precipitate [37,47]. Electrophoretic separation in zymograms is classically done under nondenaturing conditions, using either isoelectric focusing or gel electrophoresis.…”

Section: Zymogram Assaysmentioning

confidence: 99%

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Rossolini¹,

Schippa

Riccio³

et al. 1998

Cellular and Molecular Life Sciences (CMLS)

143

127

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Bacterial nonspecific acid phosphohydrolases (NSAPs) are secreted enzymes, produced as soluble periplasmic proteins or as membrane-bound lipoproteins, that are usually able to dephosphorylate a broad array of structurally unrelated substrates and exhibit optimal catalytic activity at acidic to neutral pH values. Bacterial NSAPs are monomeric or oligomeric proteins containing polypeptide components with an M(r) of 25-30 kDa. On the basis of amino acid sequence relatedness, three different molecular families of NSAPs can be distinguished, indicated as molecular class A, B and C, respectively. Members of each class share some common biophysical and functional features, but may also exhibit functional differences. NSAPs have been detected in several microbial taxa, and enzymes of different classes can be produced by the same bacterial species. Structural and phyletic relationships exist among the various bacterial NSAPs and some other bacterial and eucaryotic phosphohydrolases. Current knowledge on bacterial NSAPs is reviewed, together with analytical tools that may be useful for their characterization. An overview is also presented concerning the use of bacterial NSAPs in biotechnology.

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Section: Zymogram Assaysmentioning

confidence: 99%

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Rossolini¹,

Schippa

Riccio³

et al. 1998

Cellular and Molecular Life Sciences (CMLS)

143

127

View full text Add to dashboard Cite

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“…Thus, the two phosphatases are clearly distinct. Recently the cell-associated phosphatase of L. enzymogenes was located in the outer membrane of the organism (von Tigerstrom & Stelmaschuk, 1985).…”

Section: Introductionmentioning

confidence: 99%

“…Conditions for PAGE of the phosphatase have been described in detail (Maizel, 1971 ;von Tigerstrom, 1984). Samples were prepared under the usual denaturing conditions for the detection of protein (Fairbanks et al, 1971) and prepared in 10 mM-Tris/HCl, 1 % SDS, pH 8.0, without heating for the detection of phosphatase activity (von Tigerstrom & Stelmaschuk, 1985).…”

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of the Outer Membrane Associated Alkaline Phosphatase of Lysobacter enzymogenes

Tigerstrom¹,

Stelmaschuk²

1986

Microbiology

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The alkaline phosphatase (EC 3.1 .3.1) associated with the outer membrane of Lysobacter enzymogenes was solubilized from a crude membrane preparation with detergent and purified 219-fold using ion-exchange chromatography and gel filtration. The yield of the purified enzyme was 21 % and the specific activity was 438 units mg-I. The enzyme was most active at pH 8.5, readily hydrolysed 5'-, 2'-and 3'-ribose and deoxyribose nucleotides, glucose &phosphate, glycerophosphates and p-nitrophenylphosphate and was strongly inhibited by EDTA and 8hydroxyquinoline. The EDTA-inhibited enzyme could be reactivated to some extent by CoC12 and more effectively by ZnC12. The phosphatase was slightly activated by p-nitrophenylphosphate and from kinetic studies using this substrate two K,,, values, 0.56 x 1 0 -4~ and 3.4 x M, were estimated. The enzyme retained activity in the presence of SDS, unless it was heated, and had an apparent M , of 69000, as determined by SDS-PAGE. Gel filtration data suggested that the native enzyme might consist of at least two subunits. The properties of the enzyme were consistent with the view that it is held in the outer membrane by hydrophobic interactions. METHODS Chemicals.The sources of most of the chemicals used during the purification and characterization of the phosphatase have been reported (von Tigerstrom, 1984). In addition, DEAE-Sephacel and Sephacryl S-200 were Abbreviation: pNPP, p-nitrophenylphosphate. 0001-2825 0 1986 SGM

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“…Such large molecular sizes for OM proteins have been found previously only in Caulobacter crescentus (Agabian & Unger, 1978) and in the gliding bacteria Myxococcus xanthus (Orndorff & Dworkin, 1980) and Lysobacter enzyrnogenes (von Tigerstrom & Stelmaschuk, 1985). Major proteins in the molecular size range 30 kDa-45 kDa, characteristic of OM from most of the bacteria that have been studied (Lugtenberg & van Alphen, 1983), were absent from methanol-grown bacteria.…”

Section: Discussionmentioning

confidence: 52%

The Outer Membrane of Methylobacterium organophilum

Hancock

Williams

1986

Microbiology

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Inner and outer membranes were isolated from Methylobacterium organophilum by sucrose density centrifugation after disruption of bacteria by shaking with glass beads. The outer membrane (OM) contained all the pink oxocarotenoid pigment of the cell and unusually small amounts of phospholipid and lipopolysaccharide. An unidentified glucan was present in both membrane fractions. Several major OM proteins had molecular sizes in the range 49 kDa to 80 kDa and most of the OM proteins remained insoluble when OM or cell wall was treated with 2% (w/v) sodium dodecyl sulphate (SDS) at temperatures up to 50 "C. Neither polysaccharide nor phospholipid was solubilized under the same conditions. Increasing the concentration of methanol in the growth medium led to an increase in the bacterial phospholipid content and to increased solubility of the OM in 2% SDS. It is suggested that the resistance of the OM to solubilization by the detergent is due in part to the presence of large amounts of three unidentified polar, phosphate-free lipids that might be related to hopane polyols. Phospholipids in isolated walls and OM were rapidly degraded by endogenous phospholipase when incubated in Tris buffer at pH 8 but the unidentified lipids were retained in the particulate fraction.Abbreviations: KDO, 2-keto-3-deoxyoctonate; I M , inner membrane(s); OM, outer membrane(s).

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Localization of the Cell-associated Phosphatase in Lysobacter enzymogenes

Cited by 10 publications

References 22 publications

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Bacterial nonspecific acid phosphohydrolases: physiology, evolution and use as tools in microbial biotechnology

Purification and Characterization of the Outer Membrane Associated Alkaline Phosphatase of Lysobacter enzymogenes

The Outer Membrane of Methylobacterium organophilum

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