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.

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 Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity

1991 ◽  
Vol 275 (3) ◽  
pp. 685-688 ◽  
Author(s):  
R R Ramsay ◽  
G Mancinelli ◽  
A Arduini
Keyword(s):  
Membrane Lipids ◽  
Fatty Acyl ◽  
Carnitine Palmitoyltransferase ◽  
Membrane Properties ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Inner Membrane ◽  
Malonyl Coa ◽  
Intracellular Enzymes ◽  
Erythrocyte Plasma Membrane ◽  
Triton X

Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids.

scholarly journals Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Vivek Kumar Gupta ◽  
Rajnish Pal ◽  
Nikhat Jamal Siddiqi ◽  
Bechan Sharma
Keyword(s):  
Lead Exposure ◽  
Phosphate Buffer ◽  
Human Erythrocytes ◽  
Enzyme Substrate ◽  
The People ◽  
Occupationally Exposed ◽  
Membrane Bound ◽  
Triton X ◽  
Made In

Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte’s AChE exhibited Km for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (Ki value, 3.6 mM) which negatively influenced both the Vmax and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead.

scholarly journals Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid

1985 ◽  
Vol 228 (3) ◽  
pp. 683-688 ◽  
Author(s):  
H J Op den Camp ◽  
A Oosterhof ◽  
J H Veerkamp
Keyword(s):  
Enzyme System ◽  
Lipoteichoic Acid ◽  
Bifidobacterium Bifidum ◽  
Glycerol Phosphate ◽  
Phosphate Backbone ◽  
Membrane Bound ◽  
Triton X ◽  
Chloroform Methanol ◽  
Lipoteichoic Acids

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.

scholarly journals Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma

1975 ◽  
Vol 152 (1) ◽  
pp. 143-151 ◽  
Author(s):  
C N Murty ◽  
E Verney ◽  
H Sidransky
Keyword(s):  
Protein Synthesis ◽  
Initiation Factors ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Triton X ◽  
Stimulation Of

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.

Involvement of phospholipids in the modulation of a membrane-bound brain fucosyltransferase

1985 ◽  
Vol 63 (4) ◽  
pp. 296-304 ◽  
Author(s):  
M. Serres-Guillaumond ◽  
P. Broquet ◽  
P. Louisot
Keyword(s):  
Fatty Acids ◽  
Enzymatic Activity ◽  
Acyl Chain ◽  
Fatty Acyl ◽  
Strong Increase ◽  
Fatty Acyl Chain ◽  
Maximal Velocity ◽  
Sheep Brain ◽  
Membrane Bound ◽  
Triton X

Microsomal fucosyltransferase isolated from sheep brain is strongly enhanced by charged lysophospholipids such as lysophosphatidylinositol and lysophosphatidic acid, while the corresponding phospholipids are inhibitive. Lysophosphatidylcholine (lyso-PC) also greatly increases the enzymatic activity and leads to its solubilization. Its stimulatory effect is related to the length of the fatty acyl chain involved in the lyso-PC structure: fatty acids C18 and C20 are less activating than the fatty acids C14–C16. Stimulation is restored when C18 fatty acids are unsaturated (e.g., C18:1–C18:3). Enzymatic activity enhancement is decreased when phosphatidylcholine structures are reformed by the addition of lyso-PC and the corresponding fatty acid. The physical state of these structures has no influence. These data provide evidence that bilayer structures do not modify enzymatic activity, while micellar structures formed by detergents and lysophospholipids lead to a strong increase in fucosyltransferase activity. However, lyso-PC does not interact in exactly the same way as Triton X-100. Although they both enhance the maximal velocity of fucosyltransferase for its two substrates, GDP-fucose and asialofetuin, the effect with lyso-PC is greater, and it clearly enables a better affinity for GDP-fucose. Endogenous phospholipids are also able to modify enzymatic activity. Hydrolysis of phosphatidylcholine by phospholipase A2 leads to an enzymatic stimulation.

Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts

1996 ◽  
Vol 271 (4) ◽  
pp. G655-G663 ◽  
Author(s):  
J. T. Deng ◽  
M. F. Hoylaerts ◽  
M. E. De Broe ◽  
V. O. van Hoof
Keyword(s):  
Alkaline Phosphatase ◽  
Bile Salt ◽  
Bile Salts ◽  
Small Range ◽  
Liver Sinusoid ◽  
Gpi Anchor ◽  
Low Concentrations ◽  
Membrane Bound ◽  
Triton X

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.

scholarly journals Evidence for a novel ATP-dependent membrane-associated protease in spinach leaf mitochondria

1995 ◽  
Vol 310 (2) ◽  
pp. 527-531 ◽  
Author(s):  
C Knorpp ◽  
C Szigyarto ◽  
E Glaser
Keyword(s):  
Proteolytic Activity ◽  
Spinacia Oleracea ◽  
Plant Mitochondria ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Nicotiana Plumbaginifolia ◽  
Experimental Conditions ◽  
Spinach Leaf ◽  
Membrane Bound

We report the presence of an ATP-dependent proteolytic activity in spinach (Spinacia oleracea) leaf mitochondria. The proteolysis was observed as degradation of newly imported precursor protein. The precursor studied was that of the ATP synthase F1 beta subunit of Nicotiana plumbaginifolia, transcribed and translated in vitro. Degradation of pre-F1 beta was observed during kinetic studies of import in vitro. The degradation was characterized in chase experiments in which the precursor was imported into mitochondria. The import reaction was subsequently stopped by the addition of valinomycin and oligomycin. The fate of the imported precursor inside the mitochondria was monitored under different experimental conditions. There was no proteolytic degradation of the newly imported precursor at 15 degrees C, whereas 50% of the precursor was degraded after a 45 min incubation at 25 degrees C. The proteolytic activity was found to be ATP-dependent and was partially inhibited by a metal chelator, o-phenanthroline. Fractionation of mitochondria prior to degradation showed that all the ATP-dependent degradative activity was associated with the mitochondrial membrane fraction. The membrane-bound protease was inhibited by Pefabloc [4-(2-aminoethyl)-benzenesulphonyl fluoride hypochloride], an inhibitor of serine-type proteases and by N-ethylmaleimide, a thiol group reagent. Our studies thus describe a novel ATP-dependent membrane-associated serine-type protease in plant mitochondria that is capable of degrading newly imported non-assembled proteins.

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