scholarly journals Glycosyltransferases in the Golgi membranes of onion stem

1974 ◽  
Vol 142 (2) ◽  
pp. 203-209 ◽  
Author(s):  
Janet T. Powell ◽  
Keith Brew
Keyword(s):  
Enzyme Activities ◽  
Biological Significance ◽  
Sodium Deoxycholate ◽  
Sucrose Synthesis ◽  
Golgi Membranes ◽  
Meristematic Region ◽  
Inosine Diphosphatase ◽  
Lactose Synthesis ◽  
Cell Fractions ◽  
Triton X

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [14C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine α-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [14C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures

2002 ◽  
Vol 48 (12) ◽  
pp. 1056-1061 ◽  
Author(s):  
Daniela A Martinez ◽  
B Clara Nudel
Keyword(s):  
Enzyme Activity ◽  
Gum Arabic ◽  
Acinetobacter Calcoaceticus ◽  
Glass Beads ◽  
High Yield ◽  
Poly Ethylene Glycol ◽  
Poly Ethylene ◽  
Cell Fractions ◽  
Triton X ◽  
Effective Additive

Acinetobacter calcoaceticus BD413 produces variable amounts of an exocellular lipase that becomes rapidly inactivated upon secretion. To achieve high yield and protect the enzyme, we assayed the addition of several inert compounds to cell-free supernatants, cell fractions, and whole cultures. Glass beads, poly(ethylene glycol) 600, Triton X-100, saccharose, gum arabic, and β-cyclodextrin were among the compounds tested. β-Cyclodextrin and gum arabic (and saccharose to a lesser extent) were effective enzyme stabilizers in cell-free supernatants, while gum arabic, glass beads, and Triton X-100 improved lipase secretion from cells, and, therefore, total lipase yield (30–50%, according to the additive). In whole cultures, β-cyclodextrin was the most effective additive, particularly in combination with glass beads or gum arabic. Indeed, cultures containing β-cyclodextrin plus gum arabic were able to maintain 95% (±1.5%) of the initial lipase activity for more than 16 h, while control cultures with no additives maintained only 10% (±4%) of the enzyme activity after the same period. In conclusion, the addition of inert compounds in cultures may be considered a useful approach for achieving increased yield and lipase stabilization, amenable for downstream processing.Key words: Acinetobacter calcoaceticus, lipase, secretion, stabilization, inert additives.

scholarly journals Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system

1975 ◽  
Vol 150 (3) ◽  
pp. 537-551 ◽  
Author(s):  
P H Cooper ◽  
J N Hawthorne
Keyword(s):  
Phosphatase Activity ◽  
Metal Ion ◽  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Sodium Deoxycholate ◽  
Subcellular Fractionation ◽  
Kidney Cortex ◽  
Ph Optimum ◽  
Triton X ◽  
Hydrolysis Of

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.

scholarly journals The effect of chemical agents on the turnover of the bound phosphate associated with the sodium-and-potassium ion-stimulated adenosine triphosphatase in ox brain microsomes

1970 ◽  
Vol 120 (1) ◽  
pp. 1-13 ◽  
Author(s):  
R. Rodnight
Keyword(s):  
Sodium Chloride ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Ratio ◽  
Chemical Agents ◽  
Rapid Fall ◽  
Triton X ◽  
Hydrolysis Of ◽  
Sodium And Potassium

1. The effect of chemical agents on the turnover of the Na+-dependent bound phosphate and the simultaneous Na+-dependent hydrolysis of ATP by a membrane preparation from ox brain was studied at an ATP/protein ratio of 12.5pmol/μg. 2. The agents were added immediately after phosphorylation of the preparation in a medium containing 50mm-sodium chloride and 2.5μm-[γ-32P]ATP. 3. Concentrations of sodium chloride above 150mm, calcium chloride to 20mm and suramin to 1.4mm inhibited both phosphorylation and dephosphorylation and concomitantly slowed ATP hydrolysis. At 125mm-sodium chloride dephosphorylation and hydrolysis were slightly slowed without affecting phosphorylation. 4. Ethanol to 1.6m concentration inhibited dephosphorylation without affecting phosphorylation; the bound phosphate was increased and ATP hydrolysis slowed. 5. Ouabain to 4mm concentration partially inhibited ATP hydrolysis and caused a transient (1–2s) rise in bound phosphate followed by a rapid fall to a lower plateau value, which eventually declined to zero by the time ATP hydrolysis was complete. 6. Of the detergents examined Lubrol W, Triton X-100 and sodium deoxycholate had no significant effect on turnover. Sodium dodecyl sulphate and sodium decyl sulphate to 3.5mm and 20mm respectively completely inhibited turnover and ATP hydrolysis and stabilized the bound phosphate.

METABOLISM OF PREGNENOLONE AND PREGNENOLONE SULPHATE IN HUMAN FOETAL LIVER TISSUE IN VITRO

1974 ◽  
Vol 76 (3) ◽  
pp. 525-538 ◽  
Author(s):  
Ilpo Huhtaniemi
Keyword(s):  
Enzyme Activities ◽  
Liver Tissue ◽  
Biological Significance ◽  
Active Role ◽  
Direct Conversion ◽  
Gas Chromatography Mass Spectrometry ◽  
Conversion Rates ◽  
Significant Difference ◽  
Foetal Liver

ABSTRACT The metabolism of pregnenolone and pregnenolone sulphate was studied in incubations with minced foetal liver tissue. In most of the incubations, non-radioactive substrates were used, and the identification and quantitative determination of the metabolites formed was carried out by gasliquid chromatography and gas chromatography – mass spectrometry. Both the free and sulphated substrates were extensively metabolized by the liver preparations, and with both substrates, the same enzyme activities were observed. Furthermore, active sulphate conjugation was seen with free pregnenolone. No sulphatase activity was observed, and the direct conversion of pregnenolone sulphate to 16α-hydroxypregnenolone sulphate was demonstrated in incubations with [7-3H]pregnenolone [35S]sulphate. The highest conversion rate with both substrates, about 25 %, was for 16α-hydroxylation. Other enzyme activities detected were 20α-reduction and 3β-hydroxy-5,16-pregnadien-20-one formation. In sulphate conjugation and 16α-hydroxylation a slight decrease was observed with advancing foetal age, while in the case of 20α-reduction, somewhat higher conversion rates were observed in older foetuses. In this respect, the only significant difference between the two substrates used was the more pronounced decrease in pregnenolone sulphate 16α-hydroxylation with increasing foetal age. This study sheds more light on the biological significance of steroid sulphates as metabolic intermediates in the human foetus and further evidence for their direct conversion into other steroid sulphates was obtained. It is suggested that the foetal liver plays an active role in such conversions.

scholarly journals The distribution, abundance and subcellular localization of kinesin.

1989 ◽  
Vol 108 (6) ◽  
pp. 2335-2342 ◽  
Author(s):  
P J Hollenbeck
Keyword(s):  
Sympathetic Neurons ◽  
Molar Ratio ◽  
Soluble Form ◽  
Mitotic Spindles ◽  
Western Blots ◽  
Cultured Fibroblasts ◽  
Brij 58 ◽  
Cell Fractions ◽  
Triton X ◽  
Quantitative Immunoblotting

An antiserum which binds kinesin specifically on Western blots was used to determine the distribution and abundance of chicken kinesin by correlated immunoblotting and immunolocalization. Quantitative immunoblotting showed that the abundance of kinesin varied widely in different cell and tissue types, from 0.039% of total protein in epidermal fibroblasts to 0.309% in sympathetic neurons; of the types examined, only red blood cells lacked detectable kinesin. The molar ratio of tubulin/kinesin varied over a narrower range. To analyze the intracellular distribution of kinesin, cultured fibroblasts were fractionated by sequential extraction with saponin-, Triton X-100-, and SDS-containing buffer. Quantitative blotting of the resulting cell fractions indicated that 68% of fibroblast kinesin is in soluble form, 32% is membrane- or organelle-associated, and none is detectable in cytoskeletal fractions. To visualize this distribution, cells treated by the same extraction protocol were immunofluorescently stained with antikinesin and antitubulin. Without extraction, kinesin staining was located throughout cultured neurons and fibroblasts. However, when fibroblasts were extracted with saponin or Brij 58 before fixation, subsequent staining revealed that the remaining kinesin fraction was colocalized with interphase microtubules, but not with mitotic spindles. Prefixation extraction with Triton abolished antikinesin staining. These data suggest that kinesin may play a role in tubovesicular movement but provide no evidence for a role in mitosis.

scholarly journals Metabolism of conjugated sterols in eggplant. Part 2. Phospholipid : steryl glucoside acyltransferase.

2008 ◽  
Vol 55 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Anna Potocka ◽  
Jan Zimowski
Keyword(s):  
Divalent Cations ◽  
Solanum Melongena ◽  
Sodium Deoxycholate ◽  
Tween 20 ◽  
Acyl Donor ◽  
Kinetic Properties ◽  
Acyltransferase Activity ◽  
Steryl Glucoside ◽  
Membrane Bound ◽  
Triton X

A membrane-bound phospholipid : steryl glucoside acyltransferase from Solanum melongena leaves was partially purified and its specificity and molecular as well as kinetic properties were defined. Among the steryl glycosides tested (e.g. typical plant steryl glucosides, steryl galactosides and cholesteryl xyloside) the highest activity was found with cholesteryl glucoside, but some structurally related compounds such as sito- and stigmasteryl glucoside or galactoside as well as cholesteryl galactoside were also acylated, albeit at lower rates. The investigated enzyme was able to use all classes of phosphoglycerolipids (phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol) as an acyl source for biosynthesis of acylated steryl glucoside. Among them 1,2-dimirystoylphosphatidylic acid appeared to be the best acyl donor. Apart from phosphoglycerolipids, 1,2-diacylglycerols were also used as acyl donor for steryl glucoside acylation, although at a distinctly lower rate. The acyl moiety was transferred from the C-1 position of phospholipid molecule. The investigated acyltransferase activity was stimulated by 2-mercaptoethanol, Triton X-100, 1-monoacylglycerols and inhibited in the presence of divalent cations such as Ca(2+), Mn(2+), Zn(2+) or Co(2+), some lipids (MDGD, ceramide), detergents (Tween 20, 40, 60 and 80, Tyloxapol, sodium deoxycholate) and high ionic strength.

scholarly journals Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation

2020 ◽  
Vol 26 (3) ◽  
pp. 167-178 ◽  
Author(s):  
T T Tiemann ◽  
A M Padma ◽  
E Sehic ◽  
H Bäckdahl ◽  
M Oltean ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Vascular Perfusion ◽  
Uterus Transplantation ◽  
Live Donors ◽  
Triton X

Abstract Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

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.

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