The cell wall of Bacillus licheniformis N.C.T.C. 6346. Biosynthesis of the teichuronic acid

1. Particulate fractions prepared from disrupted cells of Bacillus licheniformis N.C.T.C. 6346 catalyse the uptake of radioactivity from UDP-[(14)C]glucuronic acid or UDP-N[(14)C]-acetylglucosamine. Maximal uptake requires the presence of both nucleotides and Mg(2+) ions. The reaction is inhibited markedly by high concentrations of novobiocin and, to a certain extent, by vancomycin and by methicillin. 2. The radioactive product formed is resistant to Pronase and is soluble in 5% (w/v) trichloroacetic acid. It is of high molecular weight, from its behaviour on columns of Sephadex G-50 or G-200, and behaves during paper electrophoresis in n-acetic acid and chromatography on DEAE-cellulose in a manner similar to teichuronic acid. 3. Both teichuronic acid and the synthesized material are resistant to testicular hyaluronidase and to Flavobacterium heparinum heparinase. 4. The specific activity of suspensions of broken cells or of washed particulate fractions is greatest when they are prepared from exponentially growing cells. Fractions obtained from late exponential-phase or stationary-phase cells have very low activity. 5. The galactosamine content of B. licheniformis N.C.T.C. 6346 cell walls increases during the exponential phase and decreases during the stationary phase.

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The purification and properties of an amino acid arylamidase from bovine milk

Canadian Journal of Biochemistry ◽

10.1139/o69-027 ◽

1969 ◽

Vol 47 (2) ◽

pp. 173-178 ◽

Cited By ~ 8

Author(s):

A. Mellors

Keyword(s):

Amino Acid ◽

Milk Fat ◽

Specific Activity ◽

Bovine Milk ◽

Deae Cellulose ◽

Maximum Activity ◽

Divalent Metal Ions ◽

Purification And Properties ◽

Milk Fat Globule ◽

High Concentrations

An amino acid arylamidase is present in bovine milk and is associated with the "microsomes" of the milk-fat globule membrane. It has been purified by DEAE-cellulose chromatography of a 0.1 M NaCl extrast of milk microsomes. The specific activity of the purified arylamidase was increased 12 700-fold over that of the milk. Three peaks of arylamidase activity could be recognized after the chromatography. One form was apparently bound to casein. The major peak of arylamidase activity hydrolyzes lysyl-, alanyl-, valyl-, and arginyl-β-naphthylamides at similar rates, with little activity against glycyl- and histidyl-β-naphthylamides. The arylamidase requires the restoration of sulfhydryl groups by dithiothreitol for maximum activity. It is inhibited by EDTA and some divalent metal ions, and only calcium ions restore the EDTA-inactivated enzyme. The optimum pH for the hydrolysis of lysyl-β-naphthylamide is pH 7.7, and high concentrations of this substrate are inhibitory.

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Inorganic pyrophosphatase of Streptococcus faecium F24

Canadian Journal of Microbiology ◽

10.1139/m72-029 ◽

1972 ◽

Vol 18 (2) ◽

pp. 183-192 ◽

Cited By ~ 1

Author(s):

Patricia R. Starr ◽

Evelyn L. Oginsky

Keyword(s):

Stationary Phase ◽

Specific Activity ◽

Deae Cellulose ◽

Inorganic Pyrophosphatase ◽

Inactivation Kinetics ◽

Starch Gel Electrophoresis ◽

Diethylaminoethyl Cellulose ◽

Single Protein ◽

Streptococcus Faecium ◽

Specific Activities

The acid Co2+-activated and the alkaline Mg2+-activated inorganic pyrophosphatases of Streptococcus faecium F24 were studied by a variety of techniques to determine whether they are activities of two distinct proteins or of a single protein. Both enzyme activities were found to be cryptic and soluble. The specific activities of both enzymes increased coordinately 1.5- to 3-fold during exponential growth at 37C, and then decreased as the cells approached stationary phase. Similar shifts in specific activity did not occur upon growth at 30C. The specific activities in extracts of cells in stationary phase varied coordinately over a 2-fold range depending on nutritional conditions. Diethylaminoethyl cellulose (DEAE-cellulose) and Sephadex G-100 column chromatography and starch–gel electrophoresis did not resolve the two activities. Slight differences in the thermal inactivation kinetics of the two activities were observed. It was concluded that the two pyrophosphatase activities are most probably those of a single protein under different assay conditions.

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A novel procedure for the rapid purification of plastocyanin from pea (Pisum sativum) leaves

Biochemical Journal ◽

10.1042/bj1930375 ◽

1981 ◽

Vol 193 (1) ◽

pp. 375-378 ◽

Cited By ~ 4

Author(s):

A R Ashton ◽

L E Anderson

Keyword(s):

Ionic Strength ◽

Pisum Sativum ◽

Deae Cellulose ◽

High Concentrations ◽

Rapid Purification ◽

Low Ionic Strength

Plastocyanin is soluble at high concentrations (greater than 3 M) of (NH4)2SO4 but under these conditions will adsorb tightly to unsubstituted Sepharose beads. This observation was utilized to purify plastocyanin from pea (Pisum sativum) in two chromatographic steps. Sepharose-bound plastocyanin was eluted with low-ionic-strength buffer and subsequently purified to homogeneity by DEAE-cellulose chromatography.

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Metabolic, Physiological, and Transcriptomics Analysis of Batch Cultures of the Green Microalga Chlamydomonas Grown on Different Acetate Concentrations

Cells ◽

10.3390/cells8111367 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1367 ◽

Cited By ~ 3

Author(s):

Bogaert ◽

Perez ◽

Rumin ◽

Giltay ◽

Carone ◽

...

Keyword(s):

Stationary Phase ◽

Biomass Yield ◽

Fatty Acid Content ◽

Biological Significance ◽

Exponential Phase ◽

Low Light ◽

Batch Mode ◽

Batch Cultures ◽

Green Microalga ◽

Transcriptomics Data

Acetate can be efficiently metabolized by the green microalga Chlamydomonas reinhardtii. The regular concentration is 17 mM, although higher concentrations are reported to increase starch and fatty acid content. To understand the responses to higher acetate concentrations, Chlamydomonas cells were cultivated in batch mode in the light at 17, 31, 44, and 57 mM acetate. Metabolic analyses show that cells grown at 57 mM acetate possess increased contents of all components analyzed (starch, chlorophylls, fatty acids, and proteins), with a three-fold increased volumetric biomass yield compared to cells cultivated at 17 mM acetate at the entry of stationary phase. Physiological analyses highlight the importance of photosynthesis for the low-acetate and exponential-phase samples. The stationary phase is reached when acetate is depleted, except for the cells grown at 57 mM acetate, which still divide until ammonium exhaustion. Surprisal analysis of the transcriptomics data supports the biological significance of our experiments. This allows the establishment of a model for acetate assimilation, its transcriptional regulation and the identification of candidates for genetic engineering of this metabolic pathway. Altogether, our analyses suggest that growing at high-acetate concentrations could increase biomass productivities in low-light and CO2-limiting air-bubbled medium for biotechnology.

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Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

Functional Plant Biology ◽

10.1071/fp05055 ◽

2005 ◽

Vol 32 (9) ◽

pp. 839

Author(s):

Rui Zhou ◽

Lailiang Cheng

Keyword(s):

Heat Treatment ◽

Thermal Stability ◽

Enzyme Activity ◽

Inorganic Phosphate ◽

Thermal Inactivation ◽

Specific Activity ◽

Apple Leaves ◽

Apparent Homogeneity ◽

Adp Glucose Pyrophosphorylase ◽

High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

Biochemical Journal ◽

10.1042/bj1620671 ◽

1977 ◽

Vol 162 (3) ◽

pp. 671-679 ◽

Cited By ~ 45

Author(s):

P S Agutter ◽

J R Harris ◽

I Stevenson

Keyword(s):

Nuclear Envelope ◽

Specific Activity ◽

Assay Medium ◽

Exogenous Rna ◽

Mammalian Liver ◽

High Concentrations ◽

Enzymic Marker ◽

Stimulation Of ◽

Nucleoside Triphosphatase

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

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Survival of virulentLegionella pneumophilain aerosols

Journal of Hygiene ◽

10.1017/s0022172400029090 ◽

1983 ◽

Vol 90 (3) ◽

pp. 451-460 ◽

Cited By ~ 54

Author(s):

P. Hambleton ◽

M. G. Broster ◽

P. J. Dennis ◽

R. Henstridge ◽

R. Fitzgeorge ◽

...

Keyword(s):

Stationary Phase ◽

Respiratory Disease ◽

Legionella Pneumophila ◽

Solid Medium ◽

Exponential Phase ◽

Metabolic Status ◽

Aqueous Suspensions

SUMMARYAqueous suspensions of virulentLegionellapneumophilagrown on solid medium retained virulence and aerosol survival characteristics for several months. Significant numbers of viable organisms were recovered from aerosols held at various relative humidities (r.h.) for up to 2 h. The organisms survived best at 65% r.h. and were least stable at 55% r.h.Exponential phase broth-grown organisms survived poorly in aerosols in comparison with stationary phase broth cultures or organisms grown on solid medium, suggesting that the metabolic status ofLegionella pneumophilaorganisms may be an important factor affecting their ability to survive in aerosols and cause respiratory disease.

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Mannose-Binding Activity of Escherichia coli: a Determinant of Attachment and Ingestion of the Bacteria by Macrophages

Infection and Immunity ◽

10.1128/iai.29.2.417-424.1980 ◽

1980 ◽

Vol 29 (2) ◽

pp. 417-424

Author(s):

Zvi Bar-Shavit ◽

Rachel Goldman ◽

Itzhak Ofek ◽

Nathan Sharon ◽

David Mirelman

Keyword(s):

Escherichia Coli ◽

Stationary Phase ◽

Pathogenic Bacteria ◽

Binding Activity ◽

Exponential Phase ◽

Restrictive Temperature ◽

Filamentous Bacteria ◽

Phagocytic Cells ◽

E Coli ◽

Mannose Binding

Recently, it was suggested that a mannose-specific lectin on the bacterial cell surface is responsible for the recognition by phagocytic cells of certain nonopsonized Escherichia coli strains. In this study we assessed the interaction of two strains of E. coli at different phases of growth with a monolayer of mouse peritoneal macrophages and developed a direct method with [ 14 C]mannan to quantitate the bacterial mannose-binding activity. Normal-sized bacteria were obtained from logarithmic and stationary phases of growth. Nonseptated filamentous cells were formed by growing the organisms in the presence of cephalexin or at a restrictive temperature. Attachment to macrophages of all bacterial forms was inhibited by methyl α- d -mannoside and mannan but not by other sugars tested. The attachment of stationary phase and filamentous bacteria to macrophages, as well as their mannose-binding activity, was similar, whereas in the exponential-phase bacteria they were markedly reduced. The results show a linear relation between the two parameters ( R = 0.98, P < 0.001). The internalization of the filamentous cells attached to macrophages during 45 min of incubation was much less efficient (20%) compared to that of exponential-phase, stationary-phase, or antibody-coated filamentous bacteria (90%). The results indicate that the mannose-binding activity of E. coli determines the recognition of the organisms by phagocytes. They further suggest that administration of β-lactam antibiotics may impair elimination of certain pathogenic bacteria by inducing the formation of filaments which are inefficiently internalized by the host's phagocytic cells.

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[Phosphotyrosine]protein phosphatase in rat brain. A major [phosphotyrosine]protein phosphatase is a 23 kDa protein distinct from acid phosphatase

Biochemical Journal ◽

10.1042/bj2390155 ◽

1986 ◽

Vol 239 (1) ◽

pp. 155-162 ◽

Cited By ~ 35

Author(s):

M Okada ◽

K Owada ◽

H Nakagawa

Keyword(s):

Acid Phosphatase ◽

Molecular Mass ◽

Rat Brain ◽

Column Chromatography ◽

Protein Phosphatase ◽

Specific Activity ◽

Deae Cellulose ◽

Sodium Vanadate ◽

Nitrophenyl Phosphate ◽

Phosphotyrosine Protein Phosphatase

A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5′-AMP, 2′-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.

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Kinetic studies on the regulation of rabbit liver pyruvate kinase

Biochemical Journal ◽

10.1042/bj1310287 ◽

1973 ◽

Vol 131 (2) ◽

pp. 287-301 ◽

Cited By ~ 33

Author(s):

M. G. Irving ◽

J. F. Williams

Keyword(s):

Pyruvate Kinase ◽

Specific Activity ◽

Deae Cellulose ◽

Kinetic Studies ◽

Rabbit Liver ◽

Hill Coefficient ◽

Saturation Curve ◽

Low Concentrations ◽

Ammonium Sulphate Fractionation ◽

The Hill

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP-, with a MgADP-/ADP2- ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH+2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km+0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+.

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