Tris-maleate buffer

2009 ◽  
Vol 2009 (2) ◽  
pp. pdb.rec11647-pdb.rec11647
Keyword(s):  
Tris Maleate

Studies on Partially Purified Pig Liver Steroid Δ4-5β-Reductase Activity

1973 ◽  
Vol 51 (12) ◽  
pp. 1661-1668 ◽  
Author(s):  
Edward J. Van Doorn ◽  
John C. Nduaguba ◽  
Albert F. Clark
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Enzyme Preparation ◽  
Reductase Activity ◽  
Enzymatic Reaction ◽  
Ph Optimum ◽  
Pig Liver ◽  
Liver Cytosol ◽  
End Products ◽  
Tris Maleate

Some properties of partially purified steroid Δ4-5β-reductase activity of pig liver cytosol have been studied using testosterone as substrate. The enzymatic activity was stable for 72 h at 4° when stored in 0.05 M Tris–maleate buffer, pH 7.4 or 8.4; storage at pH 8 at 4° resulted in a 25% decrease in activity in 30 days. The pH optimum in Tris–maleate buffers was 6.4. Enzyme activity was completely inhibited by 0.2 mM p-chloromercuribenzenesulfonate and 0.2 mM p-chloromercuribenzoate. Enzyme activity was reduced by 20% and 45% with 1.0 mM iodoacetamide and 1.0 mM N-ethylmaleimide, respectively. The end products of the enzymatic reaction, NADP+ and 5β-dihydrotestosterone, inhibited the rate of reduction of testosterone. Testosterone Δ4-5β-reductase activity was present in protein of molecular weight 25 000–30 000, as determined by gel filtration.The enzyme preparation reduced a variety of C19 and C21 steroids. The highest activity (twice that for testosterone) was found with aldosterone as substrate.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

scholarly journals Iodination of Calsequestrin in the Sarcoplasmic Reticulum of Rabbit Skeletal Muscle: A Re-Examination

1979 ◽  
Vol 32 (2) ◽  
pp. 177 ◽  
Author(s):  
Ronald K Tume
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Calcium Binding ◽  
Local Environment ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Tris Maleate ◽  
Sepharose 4B

The exposed proteins of sarcoplasmic reticulum (SR) vesicles from skeletal muscle were iodinated with the use of Sepharose 4B-bound lactoperoxidase, so that the location of the proteins in the membrane could be determined. It was found that the pattern of protein labelling could be modified simply by changing the constituents of the incubation media. This implies that the position or configuration of a particular protein in the membrane can be altered by the local environment. When the reaction was performed in the presence of 25 mM tris-maleate, pH 7 �0, alone, the Ca2+ pump ATPase (molecular weight 105000) and calsequestrin (47000) were both heavily labelled, suggesting they are at least partially exposed on the outer surface of the membrane. By contrast the high affinity calcium-binding protein (55000) was not labelled. However, when the vesicles were iodinated under conditions that were suitable for ATPase activity and Ca2+ accumulation, namely in the presence of 25 mM tris-maleate, pH 7 �0, 5 mM ATP, 5 mM Mg2+ and 0�05 mM Ca2+, a different pattern of labelling was obtained. No labelling of calsequestrin was observed whereas the extent of labelling of the Ca2+ pump ATPase remained about the same. The inclusion of anyone of the additives mentioned was effective in inhibiting the iodination of calsequestrin in the SR vesicle. When added alone, Ca2+ was more effective than Mg2+ in preventing labelling of calsequestrin. Half-maximal inhibition was observed at concentrations of approximately 0�05 mM Ca 2+ and 0�2-0�3 mM Mg2+ . Although much reduced, significant labelling of calsequestrin was observed even in the presence of 5 mM ATP. Investigations with partially purified calsequestrin revealed that the iodination of calsequestrin was the same in both the presence and absence of 1 mM Ca2 +. Therefore the reduction in label observed in intact SR vesicles probably represents a change in the location of calsequestrin within the membrane, rather than inhibition by Ca2+ of the iodination sites of the protein itself.

A simple method for ultrastructural visualization of cytoplasmic microfilament bundles (mfb) in rat hepatocytes

1978 ◽  
Vol 36 (2) ◽  
pp. 516-517
Author(s):  
S. Yokota ◽  
H.D. Fahimi
Keyword(s):  
Rat Hepatocytes ◽  
Sodium Deoxycholate ◽  
Uranyl Acetate ◽  
Cellular Motility ◽  
En Bloc ◽  
Adult Rats ◽  
Simple Method ◽  
Microfilament Bundles ◽  
Tris Maleate ◽  
Triton X

Microfilaments (MF) are present in a variety of animal and plant cells, and there is substantial evidence that they are involved in cellular motility and contractility (1). Although MF's were first discovered by electron microscopy (EM), they are not distinctly visible in routine EM preparations of whole tissue. In partially disrupted hepatocytes and in isolated bile canaliculi, however, the MF's are prominently contrasted with the uranyl acetate en bloc treatment (2). We now report our observations on the treatment of glutaraldehyde- (GA) fixed rat liver with various detergents. This procedure, followed by uranyl staining, increases markedly the electron density and the contrast of MFB's in tissue sections.Materials and Methods: Livers of adult rats were fixed by perfusion with 2.5% purified GA in 0.1M Na-cacodylate buffer, pH 7.2, and 0.05% CaCl2 for 10 min. 50-μm chopper sections were treated with 0.1-1% Triton X-100 or sodium deoxycholate (DOC) for 1 h at 25°C, rinsed briefly with the buffer, and then postfixed in 2% aqueous OsO4 for 60 min. at 4°C. This was followed by en bloc staining for 60 min. with 1% uranyl acetate in 0.2M Tris-maleate buffer, pH 5.2, and embedding in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. In control preparations the detergent treatment was omitted, but all other procedures were identical.

Phagocytosis and hydrogen peroxide production of Eosinophils in the rats spontaneously infected with Mycoplasma pulmonis

1990 ◽  
Vol 48 (3) ◽  
pp. 314-315
Author(s):  
Keiichi Moriguchi ◽  
Kei-Ichi Hirai
Keyword(s):  
Hydrogen Peroxide ◽  
Osmium Tetroxide ◽  
Male Rats ◽  
Mycoplasma Pulmonis ◽  
Infected Male ◽  
Hydrogen Peroxide Production ◽  
X Ray ◽  
Wistar Strain ◽  
Specific Pathogen ◽  
Tris Maleate

The ability of hydrogen peroxide (H2O2) production was cytochemically compared in eosinophils (EP) between specific pathogen free (SPF) and spontaneously Mycoplasma pulmonis-infected male rats (Wistar strain).Peritoneal cells including EP (PEP) were harvested with a cold Hanks' balanced salt solution containing 0.1% glucose (HBSSG). Simultaneously, blood granulocytes with eosinophils (BEP) were isolated from the same animals by a Ficoll-Hypaque method. Cells were incubated with latex particles in HBSSG for 60-90 min. Thereafter, cells were washed and transferred into a cerium (Ce) medium consisting of 0.1 M tris - maleate buffer, pH 7.5, 1mM CeCl3, 10 mM sodium azide, 0.1. glucose and 0.25 M sucrose. The incubation was carried out for 20 min at 37°C with occasional stirring. Some cells were incubated for 60 min at 37 °C in a DAB medium containing 0.1% 3.3’-diaminobenzidine 4HCl with particles in 0.1 M phosphate buffer, pH 7.4. All cells were then fixed with glutaraldehyde and osmium tetroxide before processing for electron microscopy, x-ray microanalysis of the subcellular electron-dense reaction deposits was performed under a Tracor-Northern EDX attached to a JEM-1200EX STEM system.

scholarly journals CYTOCHEMICAL DEMONSTRATION OF ORNITHINE CARBAMOYLTRANSFERASE ACTIVITY IN LIVER MITOCHONDRIA OF RAT AND MOUSE

1968 ◽  
Vol 16 (3) ◽  
pp. 172-180 ◽  
Author(s):  
AKIRA MIZUTANI
Keyword(s):  
Lead Nitrate ◽  
Liver Mitochondria ◽  
Ornithine Carbamoyltransferase ◽  
Carbamoyl Phosphate ◽  
Acid And Alkaline Phosphatase ◽  
Cytochemical Demonstration ◽  
Brush Borders ◽  
Liver And Kidney ◽  
Cold Acetone ◽  
Tris Maleate

In order to demonstrate ornithine carbamoyltransferase activity cytochemically, thin slices of liver and kidney of rat and mouse were fixed in cold acetone or formol-calcium, and incubated in a medium containing l-ornithine, carbamoyl phosphate, Tris-maleate buffer (pH 7.2), lead nitrate and sucrose. The specific reaction product occurred in the mitochondria of the hepatocytes only, and not in other cells of the liver or kidney. The specificity of the reaction was supported by the following observations. (1) The mitochondrial reaction was not obtained in sections incubated in a medium from which ornithine was omitted. (2) Other amino acids gave no reaction. (3) p-Chloromercuribenzoate suppressed the reaction. (4) The hepatocytes of chick (uricotelic) did not give the reaction. A nonspecific reaction in lysosomes and brush borders is caused probably by acid and alkaline phosphatase activities.

scholarly journals Subcellular localization of H2O2 production in human neutrophils stimulated with particles and an effect of cytochalasin-B on the cells

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 253-260 ◽  
Author(s):  
Y Ohno ◽  
K Hirai ◽  
T Kanoh ◽  
H Uchino ◽  
K Ogawa
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Cytochalasin B ◽  
Ultrastructural Localization ◽  
Human Neutrophils ◽  
H2o2 Production ◽  
Cellular Aggregation ◽  
Tris Maleate ◽  
Dense Product

Abstract The ultrastructural localization of H2O2 production in suspended polymorphonuclear leukocytes (PMN) stimulated with particles was studied using CeCl3 technique. PMN stimulated with opsonized zymosan or polystylene latex with or without IgG were incubated in 0.1 M Tris- maleate buffer with 1 mM CeCl3 and 10 mM aminotriazole. Cells were then fixed and embedded in a resin for electron microscopy. The reaction product of cerium perhydroxide was observed on the phagosomal membranes and on the areas of the plasma membrane engulfing the particles. Catalase or ferricytochrome-c decreased the deposits. p-Benzoquinone (O2- scavenger) inhibited the formation of the deposits, but KCN or NaN3 enhanced it. Pretreatment with p-diazobenzenesulfonic acid inhibited the reaction. In some PMN pretreated with cytochalasin-B, cellular aggregation was observed. The H2O2 production in these cells were observed on the membrane adherent to the particles and on the contact surface of the membrane of adjoining PMN. The plasma membrane was damaged and the electron-dense product was diffused into the cytoplasm. These results clearly show that H2O2 production is initiated at the area of the plasma membrane adherent to the particles and that H2O2 is released before the completion of phagocytosis.

scholarly journals Experimental Germ Tube Induction in Candida albicans: An Evaluation of the Effect of Sodium Bicarbonate on Morphogenesis and Comparison with Pooled Human Serum

2017 ◽  
Vol 2017 ◽  
pp. 1-5 ◽  
Author(s):  
Tapiwa Matare ◽  
Pasipanodya Nziramasanga ◽  
Lovemore Gwanzura ◽  
Valerie Robertson
Keyword(s):  
Candida Albicans ◽  
Human Serum ◽  
Germ Tube ◽  
Tube Formation ◽  
Joint Research ◽  
Significant Difference ◽  
Buffer Control ◽  
Tris Maleate ◽  
Microscope Slides ◽  
Germ Tubes

Objective. The potential of NaHCO3 versus human serum to induce germ tube formation in Candida albicans was investigated. Specimens. A total of 100 isolates were obtained from oral swabs of patients presenting with thrush. Approval for the study was granted by the Joint Research Ethics Committee (JREC/23/08). Method. Confirmed C. albicans isolates by routine methods were tested for germ tube induction using 5 different concentrations of Tris-maleate buffered NaHCO3 and Tris-maleate buffer control. Standard control strains included were C. albicans (ATCC 10231) and C. krusei (ATCC 6258). Microculture was done in 20 μL inoculums on microscope slides for 3 hours at 37°C. The rate of germ tube formation at 10-minute intervals was determined on 100 isolates using the optimum 20 mM Tris-maleate buffered NaHCO3 concentration. Parallel germ tube formation using human serum was done in test tubes. Results. The optimum concentration of NaHCO3 in Tris-maleate buffer for germ tube induction was 20 mM for 67% of isolates. Only 21% of isolates formed germ tubes in Tris-maleate buffer control. There was no significant difference in induction between human serum and Tris-maleate buffered NaHCO3. Conclusion. Tris-maleate buffered NaHCO3 induced germ tube formation in C. albicans isolates at rates similar to human serum.

scholarly journals Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

1970 ◽  
Vol 119 (4) ◽  
pp. 635-642 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Heinz Breuer
Keyword(s):  
Human Liver ◽  
Enzyme Preparation ◽  
Reaction Rate ◽  
Specific Radioactivity ◽  
Enzymic Activity ◽  
Hydroxyl Group ◽  
Freezing And Thawing ◽  
Cytosol Fraction ◽  
Tris Maleate ◽  
Conjugating Enzyme

An enzyme that conjugates the 17β-hydroxyl group of testosterone was found in the cytosol fraction of human liver. The same enzyme preparation also conjugates the 16α-hydroxyl group of oestriol. The enzymic activity could not be sedimented by centrifuging the cytosol fraction at 158000gav. for 120min. The testosterone-conjugating as well as the oestriol-conjugating activities were found in the precipitate obtained after 30% saturation of the cytosol fraction with ammonium sulphate. Filtration of the precipitate through Sephadex G-200 enriched the testosterone-conjugating enzyme 50-fold and the oestriol-conjugating enzyme 100-fold. No separation of the two activities was achieved. With labelled testosterone the product of the reaction, testosterone 17β-glucuronide, was identified by paper chromatography and by crystallization to constant specific radioactivity. Testosterone 17β-glucuronyltransferase was active between pH7.0 and 8.6 in tris–HCl and tris–maleate buffers. The apparent Km values for testosterone and UDP-glucuronic acid were 6.4 and 25μm respectively. The enzyme was active between 37 and 45°C; the activation energy was calculated to be 5kcal/mol. Oestriol did not influence the glucuronidation of testosterone. Controlled heating as well as alternate freezing and thawing of the purified enzyme preparation led to an inactivation of both testosterone-conjugating and oestriol-conjugating activities at similar rates. Testosterone and oestriol, when incubated together, gave a reaction rate that was approximately equal to the sum of the rates when the two substrates were incubated separately. The present findings suggest that testosterone and oestriol are conjugated by two separate enzymes.

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