Metabolism of phosphoglycerides in E. coli IV. The positional specificity and properties of phospbolipase A

1969 ◽  
Vol 47 (12) ◽  
pp. 1125-1128 ◽  
Author(s):  
P. Proulx ◽  
C. K. Fung
Keyword(s):  
Sodium Lauryl Sulfate ◽  
Divalent Cations ◽  
Sodium Deoxycholate ◽  
Maximal Activity ◽  
Phospholipase A ◽  
Lauryl Sulfate ◽  
Alkaline Ph ◽  
Ph Optimum ◽  
E Coli

The hydrolysis of labelled phosphatidylethanolamine by E. coli was studied in vitro. Phospholipase A, as detected by 32P-labelled lysophosphatidylethanolamine formation, had two pH optima, 5 and 8.4. On the other hand lysophosphofipase was active only in the alkaline range, had a pH optimum of 10, and was inhibited by high concentrations of either sodium deoxycholate or sodium lauryl sulfate. Phospholipase A required Ca2+ addition for maximal activity at both pH optima. Mg2+ also stimulated the activity but other divalent cations tested were slightly inhibitory or without effect. Sodium lauryl sulfate completely inhibited at pH 5. Experiments with singly and doubly labelled phosphatidylethanolamine indicated that phospholipase A1 activity was predominant at both acid and alkaline pH. Lower levels of phospholipase A2 were detectable only at alkaline pH.

scholarly journals The serum opacity reaction of Streptococcus pyogenes: general properties of the streptococcal factor and of the reaction in aged serum

1968 ◽  
Vol 66 (1) ◽  
pp. 37-47 ◽  
Author(s):  
M. J. Hill ◽  
Lewis W. Wannamaker
Keyword(s):  
Streptococcus Pyogenes ◽  
Divalent Cations ◽  
Horse Serum ◽  
Sodium Deoxycholate ◽  
Heat Stability ◽  
Maximal Activity ◽  
Free Enzyme ◽  
Ph Optimum ◽  
Serum Opacity Factor ◽  
High Concentrations

SUMMARYThe capacity of certain strains of Streptococcus pyogenes to produce opacity in aged horse serum has been studied. Cells from all stages of the growth cycle are able to produce opacity. Maximal activity is reached towards the end of the exponential phase of growth.Examination of cell fractions obtained by mechanical breakage and differential centrifugation suggested that the cell-bound activity is predominantly associated with the membrane fraction. Extraction with sodium deoxycholate yields a soluble fraction of high activity.There is considerable strain variation in heat stability of the serum opacity factor. Cell-bound activity is often quite resistant to heat, whereas extracted activity is less stable.Low concentrations of divalent cations have an activating effect, whereas high concentrations inhibit the serum opacity reaction. High concentrations of uni-valent cations are without effect on the cell-free enzyme but have an activating effect on the cell-bound enzyme.For both the cell-bound and the cell-free enzyme the pH optimum was 5·8.Although sensitive to trypsin and pepsin, the serum opacity factor appears to be resistant to streptococcal proteinase. Its activity is destroyed by formaldehyde and by periodate but is unaffected by a number of reducing agents.Pre-heating of the serum or the addition of iodoacetate did not affect the serum opacity reaction. The enhanced cholesterol esterification previously described with fresh serum appears to be a secondary reaction. Even when isolated by relatively gentle methods, α-lipoprotein serves as a substrate only in the presence of crystalline serum albumin.

The deacylation of phosphatidylinositol by rat brain preparations

1979 ◽  
Vol 57 (12) ◽  
pp. 1359-1367 ◽  
Author(s):  
T. Y. P. Shum ◽  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Rat Brain ◽  
Divalent Cations ◽  
Brain Homogenate ◽  
Phospholipase A ◽  
Ph Optimum ◽  
Major Activity ◽  
Nonionic Detergent

The deacylation of phosphatidylinositol (PI) in rat brain was studied in vitro. Using 1-acyl, 2-[1-14C-oleoyl] sn-glycerol-3-phosphoinositol and [U-14C]phosphatidylinositol as substrates, a release of 14C-free fatty acid was found when incubations were conducted with the PI labelled in the 2 position, both in the 12 000 – 106 000 g pellet (microsomal) and in the 106 000 g supernate prepared from rat brain homogenate. With the 106 000 g pellet the deacylation activity was linear with time up to 15 min and was directly proportional to the amount of protein added. Two pH optima were observed, one in the region of pH 7.5 which constituted the major activity and the other in the region of pH 6.0. The apparent Km for the enzyme activity at pH 7.5 was found to be 6.2 × 10−4 M and the Vmax was 1.24 nmol of [14C]oleic acid released per minute per milligram of protein. The Ca2+ ion stimulated the activity maximally at 5 mM while other divalent cations such as Mg2+, Mn2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, Pb2+, and Zn2+ either partially or completely inhibited the activity. The nonionic detergent, Triton X-100, stimulated the deacylation more than twofold at a concentration of 0.01%. The sulfydryl reagents, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide showed partial inhibition of the reaction which was reversed by the addition of dithiothreitol. The deacylation activity in the 106 000 g supernate from rat brain was found to be directly proportional to the amount of protein added, and to the time (up to 15 min). A pH optimum was observed in the region of pH 6.0. Substrate concentration studies showed that the apparent Km was 5.0 × 10−4 M and the Vmax was 3.93 nmol of [14C]oleic acid released per minute per milligram of protein. "lyso-PI," diacylglycerol, and fatty acid were formed at pH 6.0 and pH 7.5. The data obtained indicate that from 54 (pH 7.5) to 70% (pH 6.0) of the altered PI is due to phospholipase A2 activity, 24 (pH 6.0) to 28% (pH 7.5) is due to phospholipase "C-like" activity, and from 2 (pH 6.0) to 22% (pH 7.5) may be due to phospholipase A1 activity. These results provide evidence for the deacylation component of a deacylation–reacylation cycle for the generation of specific molecular species of PI.

AMPHOTERICIN B TOPICAL EXTEMPORANEOUS PREPARATIONS FOR THE TREATMENT OF NON-DERMATOPHYTIC ONYCHOMYCOSIS

2020 ◽  
pp. 135-139
Author(s):  
KOMESMUNEEBORIRAK PHOJANA ◽  
WERAWATGANONE PORNPEN ◽  
MUANGSIRI WALAISIRI
Keyword(s):  
Amphotericin B ◽  
Chemical Stability ◽  
High Performance ◽  
Sodium Lauryl Sulfate ◽  
Physical Stability ◽  
Lauryl Sulfate ◽  
Physical And Chemical ◽  
Injection Product

Objective: At present, the nail preparation to cure onychomycosis, caused by non-dermatophyte molds, is not commercially available in Thailand. The physical and chemical stability of amphotericin B (AmB) extemporaneous preparations in the presence of 30% dimethyl sulfoxide (DMSO) and their in vitro nail permeation was evaluated. Methods: AmB extemporaneous preparations in the presence of 30% DMSO were prepared from a commercial sterile injection product, and cream or hydrophilic ointment. Physical stability was tested at 30°C for 2 months, or using 6 heating-cooling cycles. The chemical stability and in vitro nail permeation of AmB content were analyzed using high-performance liquid chromatography (HPLC). In vitro nail permeation was performed by applying 3.5 mg/mm2 of the tested formulation on nail clippings for 5 consecutive days. Results: The AmB cream and ointment extemporaneous preparations containing 30% DMSO, a permeation enhancer, were homogeneous and pale yellow to yellow cream or ointment. The AmB ointment was stable for up to 60 days. The ointment preparation allows in vitro penetration through nails up to 14.17 μg/cm2. The ointment preparation allows significantly better penetration through than the cream preparation due to the presence of DMSO, sodium lauryl sulfate (SLS), and water in the ointment preparation. Conclusion: The AmB extemporaneous ointment was successfully compounded from a commercial sterile injection product with a beyond-use date of 60 days. The ointment preparation is currently under further investigation for in vivo efficacy.

scholarly journals Effect of Sodium Lauryl Sulfate-Fumaric Acid Coupled Addition on theIn VitroRumen Fermentation with Special Regard to Methanogenesis

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
M. A. Abdl-Rahman ◽  
F. A. R. Sawiress ◽  
A. M. Abd El-Aty
Keyword(s):  
Fumaric Acid ◽  
Sodium Lauryl Sulfate ◽  
Volatile Fatty Acids ◽  
Ph Value ◽  
Rumen Fluid ◽  
Negative Control ◽  
Gas Production ◽  
Lauryl Sulfate ◽  
Decremental Effect

The aim of the current study was to evaluate the effect of sodium lauryl sulfate-fumaric acid coupled addition onin vitromethangenesis and rumen fermentation. Evaluation was carried out usingin vitrogas production technique. Ruminal contents were collected from five steers immediately after slaughtering and used for preparation of inoculums of mixed rumen microorganisms. Rumen fluid was then mixed with the basal diet of steers and used to generate four treatments, negative control (no additives), sodium lauryl sulfate (SLS) treated, fumaric acid treated, and SLS-fumaric acid coupled addition treated. The results revealed that, relative to control, efficiency in reduction of methanogenesis was as follows: coupled addition > SLS-addition > fumaric acid addition. Both SLS-addition and SLS-fumaric acid coupled addition demonstrated a decremental effect on ammonia nitrogen (–), total short chain volatile fatty acids (SCVFAs) concentrations and the amount of substrate degraded, and an increment effect on microbial mass and microbial yield (). Nevertheless, fumaric acid did not alter any of the previously mentioned parameters but induced a decremental effect on –. Furthermore, both fumaric acid and SLS-fumaric acid coupled addition increased propionate at the expense of acetate and butyrate, while, defaunation increased acetate at the expense of propionate and butyrate. The pH value was decreased by all treatments relative to control, while, cellulase activity did not differ by different treatments. The current study can be promising strategies for suppressing ruminal methane emissions and improving ruminants feed efficiency.

scholarly journals Recombinant pseudorabies virus DNase exhibits a RecBCD-like catalytic function

1998 ◽  
Vol 330 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Chien-Yun HSIANG ◽  
Tin-Yun HO ◽  
Chin-Hui HSIANG ◽  
Tien-Jye CHANG
Keyword(s):  
Pseudorabies Virus ◽  
Nuclease Activity ◽  
Alkaline Ph ◽  
E Coli ◽  
Catalytic Function ◽  
Double Stranded Dna ◽  
Recombinant Pseudorabies Virus ◽  
Absolute Requirement ◽  
Na Ions

The pseudorabies virus (PRV) DNase gene has previously been mapped within the PRV genome. To characterize further the enzymic properties of PRV DNase, this enzyme was expressed in Escherichia coli with the use of a pET expression vector. The protein was purified to homogeneity and assayed for nuclease activity in vitro. Recombinant PRV DNase exhibited an alkaline pH preference and an absolute requirement for Mg2+ ions that could not be replaced by Ca2+ and Na+ ions. Further studies showed that PRV DNase exhibited endonuclease, 5ʹ-exonuclease and 3ʹ-exonuclease activities in both single-stranded and double-stranded DNA. This activity occurred randomly and no significant base preference was demonstrated. The multiple biochemical activities of PRV DNase are similar to the activities of Neurospora crassa endo-exonuclease and E. coli RecBCD, two additional enzymes that are involved in recombination. Taken together, the similarity of action between N. crassa endo-exonuclease, E. coli RecBCD, and PRV DNase suggests that PRV DNase might have a role in the process of recombination that occurs during PRV infection.

scholarly journals The Bacterium Thermus thermophilus, Like Hyperthermophilic Archaea, Uses a Two-Step Pathway for the Synthesis of Mannosylglycerate

2003 ◽  
Vol 69 (6) ◽  
pp. 3272-3279 ◽  
Author(s):  
Nuno Empadinhas ◽  
Luciana Albuquerque ◽  
Anke Henne ◽  
Helena Santos ◽  
Milton S. da Costa
Keyword(s):  
Biosynthetic Pathway ◽  
Divalent Cations ◽  
Thermus Thermophilus ◽  
Maximal Activity ◽  
Thermophilic Bacterium ◽  
Hyperthermophilic Archaea ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Homologous Genes ◽  
Molecular Masses

ABSTRACT The biosynthetic pathway for the synthesis of the compatible solute α-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of α-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and d-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90�C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95�C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn2+ was most effective for MPGS, while Mn2+, Co2+, Mg2+, and to a lesser extent Ni2+ activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.

Population Reductions of Gram-Negative Pathogens Following Treatments with Nisin and Chelators under Various Conditions

1995 ◽  
Vol 58 (9) ◽  
pp. 977-983 ◽  
Author(s):  
CATHERINE N. CUTTER ◽  
GREGORY R. SIRAGUSA
Keyword(s):  
Divalent Cations ◽  
Escherichia Coli O157 ◽  
Gram Negative Bacteria ◽  
Bacterial Populations ◽  
Gram Negative ◽  
E Coli ◽  
Transmission Electron ◽  
Colony Forming Units ◽  
Citrate Phosphate

When used in combination with chelating agents (EDTA, EGTA, citrate, phosphate), the bacteriocin nisin is effective for reducing populations of gram-negative bacteria in vitro. This study examined parameters (buffers, temperature presence of divalent cations) that affect nisin inhibition of Escherichia coli O157:H7 and Salmonella typhimurium. Approximately 7 log10 colony-forming units (CFU) per ml of E. coli and S. typhimurium were treated in PBS or MOPS buffers containing 50 μg/ml of purified nisin, alone or in combination with 500 mM lactate, 100 mM citrate, 50 mM EDTA, and 1% (wt/vol) sodium hexametaphosphate (pH 7.0) at 37°C for 60 min or 5°C for 30 min. Surviving bacterial populations were compared to untreated controls (buffers without nisin). Data indicated that treatments with nisin in buffers resulted in reductions of 4.30 and 2.30 log10 CFU/ml of E. coli and S. typhimurium, respectively, as compared to untreated controls. Population reductions ranging from 2.29 to 5.49 log10 CFU/ml were observed when cells were treated with nisin and chelator combinations at either 37°C for 60 min or 5°C for 30 min. The addition of magnesium and calcium to buffers with nisin decreased inhibition. Data obtained from spectrophotometric experiments indicated that treatments were causing the release of cellular constituents. However, transmission electron microscopy (TEM) analyses were inconclusive, since cellular membranes did not appear to be disrupted.

scholarly journals Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use

1998 ◽  
Vol 330 (3) ◽  
pp. 1079-1085 ◽  
Author(s):  
M. Nicholas SHAW ◽  
M. Olwen BIRCH ◽  
Andreas TINSCHERT ◽  
Veronika VENETZ ◽  
Rüdiger DIETRICH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hplc Analysis ◽  
Cell Protein ◽  
Ph Optimum ◽  
Biotin Synthase ◽  
Reaction Velocity ◽  
E Coli ◽  
Soluble Cell ◽  
A Cell

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5ʹ-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.

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