scholarly journals Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement

1983 ◽  
Vol 216 (3) ◽  
pp. 727-735
Author(s):  
Y Shidoji ◽  
C S Silverman-Jones ◽  
L M De Luca
Keyword(s):  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Phospholipase C ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Inhibitory Effect ◽  
Synthetic Activity ◽  
Dolichyl Phosphate ◽  
Liver Membranes ◽  
Endogenous Lipids

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.

scholarly journals Validation of an HPLC Method for the Simultaneous Quantification of Metabolic Reaction Products Catalysed by CYP2C11 Enzymes in Rat Liver Microsomes: In Vitro Inhibitory Effect of Salicylic Acid on CYP2C11 Enzyme

Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4294 ◽  
Author(s):  
Salhab ◽  
Naughton ◽  
Barker
Keyword(s):  
High Performance Liquid Chromatography ◽  
Enzyme Activity ◽  
Salicylic Acid ◽  
Liquid Chromatography ◽  
Rat Liver ◽  
High Performance ◽  
Inhibitory Effect ◽  
Metabolic Reaction ◽  
Rat Liver Microsomes

The inhibitory effect of new chemical entities on rat liver P450 marker activities was investigated in a functional approach towards drug development. Treatment of colorectal cancer (CRC) and chemoprevention using salicylic acid has gained a lot of attention, mainly in the prevention of the onset of colon cancer. Thus, an in vitro inhibitory effect of salicylic acid on rat CYP2C11 activity was examined by using high performance liquid chromatography (HPLC). High performance liquid chromatography analysis of a CYP2C11 assay was developed on a reversed phase C18 column (SUPELCO 25 cm × 4.6 mm × 5 µm) at 243 nm using 32% phosphate buffer (pH 3.36) and 68% methanol as a mobile phase. The CYP2C11 assay showed good linearity for all components (R2 > 0.999). Substrates and metabolites were found to be stable for up to 72 hours. Additionally, the method demonstrated good reproducibility, intra- and inter-day precision (<15%), acceptable recovery and accuracy (80%–120%), and low detection (1.3501 µM and 3.2757 µM) and quantitation limit values (4.914 µM and 9.927 µM) for 16α-hydroxytestosterone and testosterone, respectively. Salicylic acid acts reversibly as a noncompetitive (weak) inhibitor with Ki = 84.582 ± 2.67 µM (concentration of inhibitor to cause 50% inhibition of original enzyme activity (IC50) = 82.70 ± 2.67 µM) for CYP2C11 enzyme activity. This indicates a low potential to cause toxicity and drug–drug interactions.

Glycoprotein Biosynthesis: Stimulation of N-Acetylglucosaminyltransferase Activity by Cytidiee 5′-Diphosphocholine

1972 ◽  
Vol 50 (10) ◽  
pp. 1082-1093 ◽  
Author(s):  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Inhibitory Action ◽  
Inhibitory Effect ◽  
Nucleotide Sugar ◽  
Inorganic Pyrophosphate ◽  
Golgi Membranes ◽  
Cationic Detergents ◽  
Better Than ◽  
Stimulation Of

The stimulatory effect of CDP-choline on N-acetylglucosaminyltransferase activity is marked in rough microsomes but is almost absent in Golgi-rich membranes or in serum. The marked CDP-choline effect on the enzyme is evident even when the nucleotide–sugar substrate concentration is raised to near saturation. Diglyceride has an inhibitory action on the enzyme which is effectively reversed by further addition of CDP-choline. Of the other different lipid factors tested only CDP-ethanolamine has a stimulatory effect similar to CDP-choline. CDP-choline alone activates the enzyme better than Triton. CDP-choline and Triton, in different combinations of doses, show a marked synergistic effect. Cationic detergents do not activate the enzyme and inorganic pyrophosphate almost completely inhibits the enzyme activity. Phospholipase A has an inhibitory effect in the presence of CDP-choline. Phospholipase C, by itself, stimulates the enzyme activity. In the presence of CDP-choline, a higher concentration of phospholipase C partially abolishes the CDP-choline effect on the enzyme. Phosphorylcholine from labeled CDP-choline is rapidly incorporated into lecithin in the assay system used for measuring N-acetylglucosaminyltransferase activity. Capacity for lecithin synthesis is poor in Golgi membranes. However, lecithin synthesis is stimulated by adding exogenous diglyceride, but CDP-choline plus diglyceride failed to activate N-acetylglucosaminyltransferase in Golgi membranes. Finally, various possibilities have been discussed to explain the mechanism of action of CDP-choline on the enzyme.

scholarly journals Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver

1981 ◽  
Vol 91 (3) ◽  
pp. 679-688 ◽  
Author(s):  
A Ravoet ◽  
A Amar-Costesec ◽  
D Godelaine ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Golgi Complex ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Liver Microsomes ◽  
Reaction Medium ◽  
Previous Treatment ◽  
Reaction Products ◽  
Dolichyl Phosphate

To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1-phosphate derivatives is restricted to the endoplasmic reticulum in liver cells, and that the enzymes involved are similarly active in the smooth and in the rough elements.

scholarly journals Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes

1989 ◽  
Vol 259 (3) ◽  
pp. 913-916 ◽  
Author(s):  
J A Higgins ◽  
B W Hitchin ◽  
M G Low
Keyword(s):  
Endoplasmic Reticulum ◽  
Bacillus Thuringiensis ◽  
Phospholipase C ◽  
Sodium Carbonate ◽  
Plasma Membranes ◽  
Secretory Proteins ◽  
Membrane Phospholipids ◽  
Golgi Membranes ◽  
Liver Membranes ◽  
Hydrolysis Of

Phosphatidylinositol-specific phospholipase C (PI-PLC) produced by Bacillus thuringiensis has been used as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Approx. 50% of this phospholipid was hydrolysed in microsomal vesicles (endoplasmic reticulum) with no significant hydrolysis of the remaining membrane phospholipids. Latency of mannose-6-phosphatase was retained during treatment indicating that the vesicles remained impermeable. Stripping of the ribosomes did not increase hydrolysis of phosphatidylinositol; however, when the vesicles were opened using dilute sodium carbonate, hydrolysis increased to greater than 90%. Hydrolysis of phosphatidylinositol of Golgi membranes was 35% and of plasma membranes was 50%. After treatment with PI-PLC, radiolabelled secretory proteins were retained in Golgi membranes and trapped lactate dehydrogenase was retained in plasma-membrane preparations indicating that the vesicles remained closed. Hydrolysis of phosphatidylinositol increased to greater than 90% when the membranes were opened by treatment with dilute sodium carbonate. These observations indicate that PI-PLC of Bacillus thuringiensis is a suitable probe for the distribution of phosphatidylinositol in membranes, and that in liver membranes this phospholipid occurs on each side of the bilayer, a topography consistent with its diverse roles.

scholarly journals Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 541-547 ◽  
Author(s):  
K E Creek ◽  
D J Morré ◽  
C S Silverman-Jones ◽  
Y Shidoji ◽  
L M De Luca
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Subcellular Fractions ◽  
Transfer Activity ◽  
Dolichyl Phosphate ◽  
Endoplasmic Reticulum Protein ◽  
Triton X

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

scholarly journals Phospholipid content and activity of pure uridine diphosphate-glucuronyltransferase from rat liver

1978 ◽  
Vol 171 (3) ◽  
pp. 821-824 ◽  
Author(s):  
B Burchell ◽  
T Hallinan
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Rat Liver ◽  
Phospholipid Content ◽  
Uridine Diphosphate

Rat liver phospholipids were radioactively labeled in vivo before purification of UDP-glucuronyltransferase to homogeneity. The pure enzyme contained very little phospholipid (approx. 0.7 mol of phospholipid/mol of protein). The solubilization detergent Lubrol 12A9 appeared to act as a phospholipid substitute, capable of supporting UDP-glucuronyltransferase activity. Phospholipase C did not inhibit the pure enzyme activity and pure UDP-glucuronyltransferase was stimulated by 40–100% by the addition of phospholipid dispersions.

The submicrosomal distribution of dolichol and dolichol phosphokinase activity in rat liver

1982 ◽  
Vol 60 (1) ◽  
pp. 36-41 ◽  
Author(s):  
Jack W. Rip ◽  
Kenneth K. Carroll
Keyword(s):  
High Pressure ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Glycoprotein Synthesis ◽  
Dolichyl Phosphate ◽  
Glycoprotein Processing ◽  
Specific Activities ◽  
High Concentrations

Microsomes were isolated from rat liver and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER) components, and the purity of these fractions was determined. The dolichol content of each of the three fractions was estimated, using high-pressure liquid chromatography. Although highest concentrations (1940 ng/mg protein) were found in Golgi, the RER contained the largest absolute amounts. The presence of large quantities of dolichol in RER is consistent with the role of dolichol as an intermediate in asparagine-linked glycoprotein synthesis. RER and SER fractions contained high specific activities for dolichol phosphokinase, while the activity in Golgi was quite low. High concentrations of dolichol in Golgi and high dolichol phosphokinase activity in SER suggest that dolichol (and dolichyl phosphate) may be utilized in Golgi for glycoprotein processing and in the transmembrane movement of sugars such as galactose.

scholarly journals Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates

1981 ◽  
Vol 196 (1) ◽  
pp. 71-79 ◽  
Author(s):  
S Kato ◽  
M Tsuji ◽  
Y Nakanishi ◽  
S Suzuki
Keyword(s):  
Direct Effect ◽  
Rat Liver ◽  
Inhibitory Effect ◽  
Oligosaccharide Synthesis ◽  
Phenyl Phosphate ◽  
Dolichyl Phosphate ◽  
Exogenous Substrate ◽  
Nitrophenyl Phosphate ◽  
The Individual ◽  
Related Compounds

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.

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