scholarly journals The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous

1982 ◽  
Vol 201 (3) ◽  
pp. 515-521 ◽  
Author(s):  
P S J Cheetham ◽  
P Dunnill ◽  
M D Lilly
Keyword(s):  
Cell Membrane ◽  
Membrane Lipids ◽  
Cholesterol Oxidase ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Solubilized Enzyme ◽  
High Concentrations ◽  
Triton X ◽  
Catalytic Functions ◽  
Hydrophobic Anchor

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.

scholarly journals Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes

1993 ◽  
Vol 289 (3) ◽  
pp. 777-782 ◽  
Author(s):  
H Schmidt ◽  
E Heinz
Keyword(s):  
Double Bond ◽  
Spinacia Oleracea ◽  
Membrane Lipids ◽  
Fatty Acyl ◽  
Acyl Residue ◽  
Solubilized Enzyme ◽  
Membrane Bound ◽  
Triton X ◽  
Envelope Membranes

In plants, polyenoic fatty acids are synthesized by desaturase enzymes which use acyl groups of membrane lipids as substrates. To provide direct ‘in vitro’ evidence for this reaction, we solubilized envelope membranes from spinach (Spinacia oleracea) chloroplasts with Triton X-100 to release a membrane-bound n-6 desaturase. In the presence of oxygen and reduced ferredoxin, the solubilized enzyme desaturated a variety of substrates, such as free oleic acid, free erucic acid, 1-oleoyl-sn-glycerol 3-phosphate and the three galactolipids 1-oleoyl-2-(7′-cis-hexadecenoyl)-3-beta-D-galactopyranosyl-sn-glycerol, 1,2-dioleoyl-3-beta-D-galactopyranosyl-sn-glycerol and the ether analogue 1,2-di-(9′-cis-octadecenyl)-3-beta-D-galactopyranosyl-sn- glycerol. The in vitro desaturation of these exogenously added complex lipids with ester- and ether-linked substrate chains is unambiguous evidence for lipid-linked desaturation. The enzyme measures the insertion of the new double bond from the methyl end and the existing (n-9)-cis-double bond of an appropriate acyl or alkyl chain. The distal part of the substrate group, normally the carboxy end of a fatty acyl residue, is of less importance and, in particular, its activation in thioester form is not required.

Interaction of Different Extracts ofPrimula heterochromaStapf. with Red Blood Cell Membrane Lipids and Proteins: Antioxidant and Antihemolytic Effects

2012 ◽  
Vol 9 (4) ◽  
pp. 285-292 ◽  
Author(s):  
Seyed Mohammad Nabavi ◽  
Seyed Fazel Nabavi ◽  
William N. Setzer ◽  
Heshmatollah Alinezhad ◽  
Mahboobeh Zare ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Membrane Lipids ◽  
Red Blood Cell Membrane

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

1987 ◽  
Vol 65 (1) ◽  
pp. 8-18 ◽  
Author(s):  
Rex K. M. Wong ◽  
Christine P. Nichol ◽  
M. Chandra Sekar ◽  
Basil D. Roufogalis
Keyword(s):  
Chain Length ◽  
Membrane Lipids ◽  
Acetylcholinesterase Activity ◽  
Exchange Chromatography ◽  
Erythrocyte Membranes ◽  
Tween 20 ◽  
Bovine Erythrocyte ◽  
Critical Micelle Concentrations ◽  
Triton X ◽  
Nonionic Detergents

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octy1 β-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70–80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

scholarly journals Acetylcholinesterase aus Rindererythrozyten. Reinigung und Eigenschaften des mit und ohne Triton X-100 solubilisierten Enzyms / Acetylcholinesterase from Bovine Erythrocytes. Purification and Properties of the En­zyme Solubilized in the Presence and the Absence of Triton X-100

1979 ◽  
Vol 34 (9-10) ◽  
pp. 721-725 ◽  
Author(s):  
Heinz Großmann ◽  
Manfred Liefländer
Keyword(s):  
Amino Acid ◽  
Specific Activity ◽  
Multiple Forms ◽  
Terminal Amino Acid ◽  
Enzyme Preparations ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Terminal Amino ◽  
Triton X ◽  
Bovine Erythrocytes

Abstract Acetylcholinesterase was released from bovine erythrocytes by Triton X-100 treatment and pu­rified by twofold affinity chromatography. The detergentfree enzyme was obtained with a specific activity of 4130 U /mg (303 000-fold purification) and a 25% yield. Alternatively, the commercial available crude enzyme was purified. The latter preparation has an uniform molecular weight (Mr 175 000). The Triton-solubilized enzyme, however, can be resolved after removal of the detergent in eight multiple forms (Mr 175 000 and multiple values), in the presence of Triton there exists only one form (Mr 338 000). The amino acid composition of the two enzyme preparations differs significantly. No differences were observed with respect to other properties: SDS gel electrophore­sis revealed two protein bands (Mr 166 000 and 86 000) with both preparations. The enzyme is a glycoprotein with a pI value of 4.3 and contains strongly bound phosphatidylethanolamine. The N-terminal amino acid has been found to be Glu (or Gin).

scholarly journals Insights into the Mode of Action of Chitosan as an Antibacterial Compound

2008 ◽  
Vol 74 (12) ◽  
pp. 3764-3773 ◽  
Author(s):  
Dina Raafat ◽  
Kristine von Bargen ◽  
Albert Haas ◽  
Hans-Georg Sahl
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
Cell Membrane ◽  
Mode Of Action ◽  
Membrane Lipids ◽  
Expression Profiles ◽  
Transcriptional Response ◽  
Response Analysis ◽  
Sequence Of Events ◽  
Wide Range

ABSTRACT Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Effect of herbicides on plant cell membrane lipids

1979 ◽  
pp. 45-76 ◽  
Author(s):  
C. M. Rivera ◽  
Donald Penner
Keyword(s):  
Cell Membrane ◽  
Plant Cell ◽  
Membrane Lipids

Reaktionen der Hydrogenase aus Rhodopseudomonas capsulata im partikelgebundenen und gelösten Zustand

1969 ◽  
Vol 24 (5) ◽  
pp. 603-612 ◽  
Author(s):  
J.-H. Klemme
Keyword(s):  
Cytochrome C ◽  
Ammonium Sulfate ◽  
Deae Cellulose ◽  
Reaction Rates ◽  
Rhodopseudomonas Capsulata ◽  
Hydrogenase Activity ◽  
Original Activity ◽  
Dispersing Agent ◽  
Solubilized Enzyme ◽  
Triton X

In cell free extracts of Rps. capsulata obtained by exposure of cells to ultrasonic oscillation, about 90% of the hydrogenase is associated with the particulate chromatophore fraction. The particulate enzyme reacts with methylene blue (MB), menadione, phenazonium methosulfate (PMS), dichlorophenolindophenol (DCPIP), cytochrome c, p-benzoquinone (BQ), ferricyanide and O2,, but does not react with benzylviologen (BV), pyridinnucleotides and flavinnucleotides. Treatment of chromatophores with sodiumlaurylsulfate inactivates the hydrogenase reaction with PMS, DCPIP, BQ and ferricyanide. The MB-linked or menadione-linked hydrogenase is not destroyed by the detergent. The hydrogenase reaction with BV is increased more than 20-fold after incubation of the chromatophores with the lipid-dispersing agent. Treatment of chromatophores with acetone and petroleum ether almost completely inactivates the hydrogenase reaction with PMS and BQ. The reaction rate of the DCPIP-linked and the ferricyanide-linked hydrogenase is somewhat decreased, whereas the MB-linked, the menadione-linked and the BV-linked hydrogenase reactions still exhibit about 100% of the original activity. By extraction of the acetone-treated chromatophores with glycine-NaOH-buffer (pH 9), about 10 — 15% of the particulate hydrogenase is solubilized. The enzyme was 9-fold purified by ammonium sulfate precipitation and chromatography on DEAE-cellulose. The purified enzyme contains no cytochrome. The relative reaction rates of the solubilized enzyme with different electron acceptors are similar to the corresponding reaction rates of the acetonetreated chromatophores. Extraction of chromatophores with n-butanol results in the solubilization of 5 — 10% of the particulate enzyme. By extraction of acetone-treated chromatophores with 0,5% Triton X-100, 40% of the particulate hydrogenase is solubilized. The fractionation of the extract with ammonium sulfate results in the isolation of a cytochrome c-containing particle which exhibits a 3-fold increased hydrogenase activity.

scholarly journals Amino Acid Substitutions in Positions 385 and 393 of the Hydrophobic Region of VP4 May Be Associated with Rotavirus Attenuation and Cell Culture Adaptation

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 408
Author(s):  
Yusheng Guo ◽  
David E. Wentworth ◽  
Karla M. Stucker ◽  
Rebecca A. Halpin ◽  
Ham Ching Lam ◽  
...  
Keyword(s):  
Cell Culture ◽  
Amino Acid ◽  
Reverse Genetics ◽  
Vaccine Development ◽  
Nonsynonymous Substitution ◽  
Amino Acid Substitutions ◽  
Viral Gastroenteritis ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Underlying Mechanisms

Rotaviruses (RVs) are the leading cause of the acute viral gastroenteritis in young children and livestock animals worldwide. Although live attenuated vaccines have been applied to control RV infection for many years, the underlying mechanisms of RV attenuation following cell culture adaption are unknown. To study these mechanisms at the genomic level, we have sequenced and conducted a comparative analysis of two virulent human (Wa, G1P[8] and M, G3P[8]) and two virulent porcine (Gottfried, G4P[6] and OSU, G5P[7]) RV strains maintained in gnotobiotic piglets for 22, 11, 12 and 9 serial passages, respectively, with their attenuated counterparts serially passaged in MA-104 cell cultures for 25, 43, 54 and 43 passages, respectively. We showed that most of the mutations were clustered in the VP4 gene, with a relatively high nonsynonymous substitution rate (81.2%). Moreover, two amino acid substitutions observed in the VP4 gene were conserved between two or more strain pairs. D385N substitution was found in M, Wa and Gottfried strains, and another one, S471H/L was present in Wa and Gottfried strains. Importantly, D385 was reported previously in another study and may be involved in regulation of virus entry. Of interest, although no 385 substitution was found in OSU strains, the attenuated OSU strain contained a unique D393H substitution within the same VP4 hydrophobic domain. Collectively, our data suggest that the VP4 hydrophobic region may play an important role in RV attenuation and aa385 and aa393 may represent potential targets for RV vaccine development using reverse genetics and site-specific mutagenesis.

Different Protein-Lipid Interaction in Human Red Blood Cell Membrane of Rh Positive and Rh Negative Blood Compared with Rhnull

1981 ◽  
Vol 36 (11-12) ◽  
pp. 988-996 ◽  
Author(s):  
Dietmar Dorn-Zachertz ◽  
Guido Zimmer
Keyword(s):  
Cell Membrane ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Disulfide Bonds ◽  
Membrane Lipids ◽  
Infrared Spectrometry ◽  
Red Blood Cell Membrane ◽  
Fluorescence Measurements ◽  
Upper Level ◽  
Lipid Interaction

Abstract 1-anilino-naphthalene-8 -sulfonate (ANS) fluorescence measurements have revealed that red blood cell membrane of the Rhnull type undergoes a transition at about 16 °C. In contrast, viscosity measurements of the extracted membrane lipids showed the usually observed transition at about 18 °C. Lower values of titratable sulfhydryl (SH) groups were observed in Rhnull membrane using 5,5′-dithiobis-(2-nitro-benzoic-acid) (Nbs2). In contrast, disulfide bonds in Rhnull membrane were estimated to be about 3 times the value of the controls. Spin labeling experiments using 2-(3-carboxypropyl)-4, 4 dimethyl-2-tridecyl 3-oxazolidinyl-oxyl were carried out with phospholipase A2 modified membranes. The mobile part of the spectra was significantly increased on the Rhnull membrane. In the presence of ᴅ-glucose, infrared spectrometry showed a larger reduction of the intensity of the POO-band in Rhnull membrane. In contrast to controls, binding of the reagent diethylpyrocarbonate resulted in no significant changes of the Rhnull membrane as determined by electron spin resonance (ESR) measurements. ᴅ-glucose transport activity was found to be at the upper level of a group of Rh positive and Rh negative persons. It is suggested that the intensity of the polar protein-lipid interaction is reduced in Rhnull membrane.

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