Purification and characterization of Pseudomonas aeruginosa alkaline phosphatase

1973 ◽  
Vol 19 (10) ◽  
pp. 1225-1233 ◽  
Author(s):  
D. F. Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Sodium Dodecyl ◽  
Current Theory ◽  
Outer Cell ◽  
Ph Optimum ◽  
Nitrophenyl Phosphate ◽  
A Subunit

Alkaline phosphatase and a subunit form of the enzyme have been isolated from Pseudomonas aeruginosa. The enzyme is pure as judged by molecular-sieve chromatography, sodium dodecyl gel electrophoresis, and ultracentrifugation. The enzyme possesses the following properties: (a) existence of three forms: monomer mol. wt. 39 000, dimer mol. wt. 68 000, and tetramer mol. wt. 139 000; (b) pH optimum 10.5; (c) Michaelis constant Km = 6.6 × 10−5 M p-nitrophenyl phosphate; and (d) energy of activation 5647 cal/mol. Amino acid analysis indicates a protein that is hydrophobic. Its physical behavior in solution supports this conclusion. These results explain the observed association of alkaline phosphatase and lipopolysaccharide and substantiate the current theory that the alkaline phosphatase of P. aeruginosa is bound to the outer cell wall in vivo.

An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

1978 ◽  
Vol 24 (4) ◽  
pp. 427-432 ◽  
Author(s):  
M. L. Marceau-Day ◽  
D. F Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Alkaline Phosphatase ◽  
Core Region ◽  
Significant Activity ◽  
Wild Type ◽  
Glycerol Phosphate ◽  
The Core ◽  
Nitrophenyl Phosphate ◽  
Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase

1980 ◽  
Vol 238 (1) ◽  
pp. G3-G9 ◽  
Author(s):  
M. H. Humphreys ◽  
G. A. Kaysen ◽  
L. Y. Chou ◽  
J. B. Watson
Keyword(s):  
Alkaline Phosphatase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intestinal Alkaline Phosphatase ◽  
Ph Optimum ◽  
Anion Exchange Column ◽  
Calf Intestinal Alkaline Phosphatase ◽  
Commercial Preparation ◽  
Nitrophenyl Phosphate ◽  
Single Protein

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.

Genetic analysis of laminin A reveals diverse functions during morphogenesis in Drosophila

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 325-337 ◽  
Author(s):  
C. Henchcliffe ◽  
L. Garcia-Alonso ◽  
J. Tang ◽  
C.S. Goodman
Keyword(s):  
Cell Fate ◽  
Genetic Characterization ◽  
Complete Loss ◽  
Loss Of Function ◽  
Partial Loss ◽  
Multidomain Structure ◽  
A Subunit ◽  
Wing Structure

In order to dissect the functions of laminin A in vivo, we have undertaken a molecular and genetic characterization of the laminin A subunit (lamA) gene in Drosophila. Sequence analysis predicts a multidomain structure similar to mammalian homologs. We generated a series of complete and partial loss-of-function mutant alleles of the lamA gene; complete loss-of-function mutations lead to late embryonic lethality. Certain combinations of partial loss-of-function lamA alleles give rise to escaper adults, which have rough eyes associated with changes in cell fate and pattern, misshapen legs and defects in wing structure. These phenotypes suggest that laminin A has diverse functions during morphogenesis in Drosophila.

Molecular mechanisms driving the in vivo development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR Pseudomonas aeruginosa infections

2020 ◽  
Vol 76 (1) ◽  
pp. 91-100
Author(s):  
Jorge Arca-Suárez ◽  
Cristina Lasarte-Monterrubio ◽  
Bruno-Kotska Rodiño-Janeiro ◽  
Gabriel Cabot ◽  
Juan Carlos Vázquez-Ucha ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Genomic Analysis ◽  
Resistance Mechanisms ◽  
Comparative Genomic ◽  
Development Of Resistance ◽  
Structural Impact ◽  
Genetic Context

Abstract Background The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning. Objectives Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections. Methods Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes. Results The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold. Conclusions Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa.

scholarly journals Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

2012 ◽  
Vol 78 (17) ◽  
pp. 6161-6171 ◽  
Author(s):  
Christoph Sygmund ◽  
Daniel Kracher ◽  
Stefan Scheiblbrandner ◽  
Kawah Zahma ◽  
Alfons K. G. Felice ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Enzyme System ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Carbohydrate Binding ◽  
Ph Optimum ◽  
Content Type ◽  
Polysaccharide Monooxygenase

ABSTRACTThe genome ofNeurospora crassaencodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome ofN. crassaand preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced inPichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochromec, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (kcatandKmvalues) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the hemebcofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) fromN. crassawas expressed inP. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

scholarly journals THE SEPARATION AND CHARACTERIZATION OF SUBMICROSCOPIC COMPONENTS OF HEPATIC CYTOPLASM

1959 ◽  
Vol 7 (2) ◽  
pp. 126-132 ◽  
Author(s):  
MITURU TAKANAMI
Keyword(s):  
Alkaline Phosphatase ◽  
Principal Components ◽  
Biochemical Properties ◽  
Sedimentation Rates ◽  
Differential Centrifugation ◽  
Ultrasonic Oscillation ◽  
Ribonucleoprotein Complexes ◽  
Acid And Alkaline Phosphatase

In an ultracentrifugal study on the cytoplasmic supernatant of rabbit liver, the following two principal components were separated from the supernatant by differential centrifugation and their biochemical properties investigated: (1) a granular substance sedimented at a rate of about 250s (250s component) and (2) a few macromolecular components the sedimentation rates of which were roughly in the range of from 40s to 100s (macromolecular components). The 250s component, which was rich in lipids and easily disintegrated into smaller units by treatments of ultrasonic oscillation and of Nadesoxycholate, exhibited much higher activities of dipeptidase, acid and alkaline phosphatase than the macromolecular components. By contrast, the latter macromolecular components which belonged to ribonucleoprotein complexes exhibited comparatively high activities of RNase and esterase. Uptake in vivo of radioactive phosphate (P32) by the RNA contained in the above two principal components markedly differed from each other. When the RNA contained in the 250s component was separated by the use of Nadesoxycholate into RNA in a non-sedimentable portion and that in a sedimentable portion corresponding to a ribonucleoprotein coplex, the RNA in the latter state showed an uptake rate extremely different from that of the macromolecular components. So it is emphasized that the ribonucleoprotein complex comprised in the 250s component and that existing free in the cytoplasm (i.e. macromolecular components) are metabolically different.

Isolation and characterization of a FAD-dependent NADH diaphorase from Methanospirillum hungatei strain GP1

1981 ◽  
Vol 59 (2) ◽  
pp. 83-91 ◽  
Author(s):  
R. C. McKellar ◽  
K. M. Shaw ◽  
G. D. Sprott
Keyword(s):  
Gel Electrophoresis ◽  
Superoxide Anion ◽  
Chelating Agents ◽  
Sodium Dodecyl ◽  
Sulfhydryl Reagents ◽  
Ph Optimum ◽  
Methanospirillum Hungatei ◽  
Isolation And Characterization ◽  
Nadh Diaphorase

Crude extracts of Methanospirillum hungatei strain GP1 contained NADH and NADPH diaphorase activities. After a 483-fold purification of the NADH diaphorase the enzyme was further separated from contaminating proteins by polyacrylamide disc gel electrophoresis. Two distinct activity bands were extracted from the acrylamide, each one having oxygen, 2,6-dichlorophenoiindophenol, and cytochrome c linked activities. In these preparations NADPH could not replace NADH as electron donor. During the initial purification steps all activity was lost due to the removal of a readily released cofactor. Enzyme activity was restored by either FAD or a FAD fraction isolated from M. hungatei. Oxidase activity exhibited a broad pH optimum from 7.0 to 8.5 and apparent Km values of 26 μM for NADH and 0.2 μM for FAD. Superoxide anion, formed in the presence of oxygen, accounted for all of the NADH consumed in this reaction. The molecular weight of the diaphorase was about 117 500 by sodium dodecyl sulfate gel electrophoresis. Sulfhydryl reagents and chelating agents were inhibitory. Inactivation, which occurred during storage in phosphate buffer at 4 °C, was delayed by dithiothreitol. The isolated NADH diaphorase lacked NADPH:NAD transhydrogenase and NAD reductase activities.

scholarly journals The identification and characterization of two separate carboxylesterases in guinea-pig serum

1983 ◽  
Vol 215 (1) ◽  
pp. 91-99 ◽  
Author(s):  
K Cain ◽  
E Reiner ◽  
D G Williams
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Selective Inhibitor ◽  
Nitrophenyl Phosphate ◽  
Phosphate Treatment ◽  
Pig Serum

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.

scholarly journals Identification and characterization of an iron-regulated gene, chtA, required for the utilization of the xenosiderophores aerobactin, rhizobactin 1021 and schizokinen by Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 945-954 ◽  
Author(s):  
Páraic Ó Cuív ◽  
Paul Clarke ◽  
Michael O'Connell
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Membrane Receptor ◽  
Outer Membrane Receptor ◽  
Significant Similarity ◽  
Hydroxamate Siderophores ◽  
Insertional Inactivation ◽  
Titration Assay

Pseudomonas aeruginosa utilizes several xenosiderophores under conditions of iron limitation, including the citrate hydroxamate siderophore aerobactin. Analysis of the P. aeruginosa genome sequence revealed the presence of two genes, chtA (PA4675) and PA1365, encoding proteins displaying significant similarity to the aerobactin outer-membrane receptor, IutA, of Escherichia coli. The chtA and PA1365 genes were mutated by insertional inactivation and it was demonstrated that ChtA is the outer-membrane receptor for aerobactin. ChtA also mediated the utilization of rhizobactin 1021 and schizokinen, which are structurally similar to aerobactin. In contrast to the utilization of other xenosiderophores by P. aeruginosa, there was no apparent redundancy in the utilization of aerobactin, rhizobactin 1021 and schizokinen. The utilization of citrate hydroxamate siderophores by P. aeruginosa was demonstrated to be TonB1 dependent. A Fur box was identified in the region directly upstream of chtA and it was demonstrated by the in vivo Fur titration assay that this region is capable of binding Fur and accordingly that expression of chtA is iron regulated. The PA1365 mutant was unaffected in the utilization of citrate hydroxamate siderophores.

scholarly journals Functional Characterization of a Serine/Threonine Protein Kinase of Pseudomonas aeruginosa

1999 ◽  
Vol 67 (10) ◽  
pp. 5386-5394 ◽  
Author(s):  
S. Timothy Motley ◽  
Stephen Lory
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid ◽  
Migration Behavior

ABSTRACT Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in Pseudomonas aeruginosa which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The ppkA gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in P. aeruginosa, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.

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