scholarly journals Dolichyl sulphate and H-phosphonate: enzymatic reactions with activated sugars.

1998 ◽  
Vol 45 (4) ◽  
pp. 1021-1030
Author(s):  
O V Sizova ◽  
S D Maltsev ◽  
V N Shibaev ◽  
W J Jankowski ◽  
T Chojnacki
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Lower Activity ◽  
Enzymatic Reactions ◽  
Rat Liver Microsomes ◽  
Reaction Products ◽  
Microsomal Enzymes ◽  
Dolichyl Phosphate

Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.

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.

Dolichyl phosphate phosphatase in rat liver microsomes

1984 ◽  
Vol 794 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Daniel O. Boscoboinik ◽  
Silvia Morera ◽  
Enrique Belocopitow
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Dolichyl Phosphate

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

1980 ◽  
Vol 58 (10) ◽  
pp. 1051-1056 ◽  
Author(s):  
Jack W. Rip ◽  
Kenneth K. Carroll
Keyword(s):  
Rat Liver ◽  
Metal Ion ◽  
Reaction Rate ◽  
Liver Microsomes ◽  
Microsomal Protein ◽  
Optimum Concentration ◽  
Nucleoside Triphosphate ◽  
Rat Liver Microsomes ◽  
Detergent Concentration ◽  
Dolichyl Phosphate

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 μM). An apparent Km of 50 μM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2− is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10–15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 μM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.

scholarly journals Rat liver microsomes catalyse mannosyl transfer from GDP-d-mannose to retinyl phosphate with high efficiency in the absence of detergents

1981 ◽  
Vol 200 (3) ◽  
pp. 529-538 ◽  
Author(s):  
Yoshihiro Shidoji ◽  
Luigi M. De Luca
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Rat Liver ◽  
Bovine Serum ◽  
High Efficiency ◽  
Liver Microsomes ◽  
Water Soluble ◽  
Rat Liver Microsomes ◽  
Uptake System ◽  
Dolichyl Phosphate

In the absence of detergent, the transfer of mannose from GDP-mannose to rat liver microsomal vesicles was highly stimulated by exogenous retinyl phosphate in incubations containing bovine serum albumin, as measured in a filter binding assay. Under these conditions 65% of mannose 6-phosphatase activity was latent. The transfer process was linear with time up to 5min and with protein concentration up to 1.5mg/0.2ml. It was also temperature-dependent. The microsomal uptake of mannose was highly dependent on retinyl phosphate and was saturable against increasing amounts of retinyl phosphate, a concentration of 15μm giving half-maximal transfer. The uptake system was also saturated by increasing concentrations of GDP-mannose, with an apparent Km of 18μm. Neither exogenous dolichyl phosphate nor non-phosphorylated retinoids were active in this process in the absence of detergent. Phosphatidylethanolamine and synthetic dipalmitoylglycerophosphocholine were also without activity. Several water-soluble organic phosphates (1.5mm), such as phenyl phosphate, 4-nitrophenyl phosphate, phosphoserine and phosphocholine, did not inhibit the retinyl phosphate-stimulated mannosyl transfer to microsomes. This mannosyl-transfer activity was highest in microsomes and marginal in mitochondria, plasma and nuclear membranes. It was specific for mannose residues from GDP-mannose and did not occur with UDP-[3H]galactose, UDP- or GDP-[14C]glucose, UDP-N-acetyl[14C]-glucosamine and UDP-N-acetyl[14C]galactosamine, all at 24μm. The mannosyl transfer was inhibited 85% by 3mm-EDTA and 93% by 0.8mm-amphomycin. At 2min, 90% of the radioactivity retained on the filter could be extracted with chloroform/methanol (2:1, v/v) and mainly co-migrated with retinyl phosphate mannose by t.l.c. This mannolipid was shown to bind to immunoglobulin G fraction of anti-(vitamin A) serum and was displaced by a large excess of retinoic acid, thus confirming the presence of the β-ionone ring in the mannolipid. The amount of retinyl phosphate mannose formed in the bovine serum albumin/retinyl phosphate incubation is about 100-fold greater than in incubations containing 0.5% Triton X-100. In contrast with the lack of activity as a mannosyl acceptor for exogenous dolichyl phosphate in the present assay system, endogenous dolichyl phosphate clearly functions as an acceptor. Moreover in the same incubations a mannolipid with chromatographic properties of retinyl phosphate mannose was also synthesized from endogenous lipid acceptor. The biosynthesis of this mannolipid (retinyl phosphate mannose) was optimal at MnCl2 concentrations between 5 and 10mm and could not be detected below 0.6mm-MnCl2, when synthesis of dolichyl phosphate mannose from endogenous dolichyl phosphate was about 80% of optimal synthesis. Under optimal conditions (5mm-MnCl2) endogenous retinyl phosphate mannose represented about 20% of dolichyl phosphate mannose at 15min of incubation at 37°C.

Dolichol and polyprenol kinase activities in microsomes from etiolated rye seedlings

1992 ◽  
Vol 70 (6) ◽  
pp. 455-459 ◽  
Author(s):  
Robert T. Rymerson ◽  
Kenneth K. Carroll ◽  
Jack W. Rip
Keyword(s):  
Rat Liver ◽  
Divalent Cations ◽  
Liver Microsomes ◽  
Microsomal Protein ◽  
Rat Liver Microsomes ◽  
Ph Optimum ◽  
Dolichyl Phosphate ◽  
Triton X ◽  
Enhance Activity ◽  
Better Than

Dolichol kinase activity in microsomes from etiolated rye seedlings had a pH optimum at 8 with a shoulder at pH 6.5. Triton X-100 (0.4%) was required for optimum activity. Exogenous divalent cations did not enhance activity, although Mg+2 was added routinely. Rye microsomes were found to contain dolichol and polyprenol in a ratio of 3 to 2. Rye, soybean embryo, and rat liver microsomes catalyzed the synthesis of 78, 52, and 516 nmol [14C]dolichyl phosphate/(mg microsomal protein∙h) compared with 21, 22, and 49 nmol [3H]polyprenyl phosphate/(mg microsomal protein∙h), respectively. It is clear that microsomes from plant systems can catalyze the phosphorylation of polyprenol better than rat liver when compared with their abilities to catalyze the phosphorylation of dolichol. It is not known whether one or more kinases is responsible for catalyzing the phosphorylation of these two closely related groups of compounds.Key words: dolichol, polyprenol, dolichyl phosphate, polyprenyl phosphate, kinases.

Metabolic Analysis of Triptolide Microspheres in Human, Dog, Rabbit and Rat Liver Microsomes with UPLC-MS/MS Method

2020 ◽  
Vol 17 ◽  
Author(s):  
LiJuan Wang ◽  
Yan Liu ◽  
Rui Li ◽  
DongXian He
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Internal Standard ◽  
Gradient Elution ◽  
Rat Liver Microsomes ◽  
Good Precision ◽  
Kinetics Parameters ◽  
Measure Concentration ◽  
Standard Curve ◽  
Variation Of Parameters

Objectives: Triptolide (TPL) has been shown to have a good clinical effect on rheumatoid arthritis (RA). We designed TPL microspheres (TPL-MS) and investigated its metabolic behavior in human, dog, rabbit and rat liver microsomes (HLM, DLM, RLM and SDRLM) with UPLC-MS/MS method. Methods: First, a UPLC-MS/MS method was established to measure concentration of TPL in samples. The sample was separated on a C18 column (2.1×100 mm, 1.8μm) and eluted with a gradient elution. The precursor ion/product ion were m/z 378.1/361.0 for TPL and 260.0/116.2 for the internal standard. Then T1/2, Vmax and CLint were calculated from the above data. Finally, the metabolites of TPL-MS were identified by high-resolution UPLC-MS/MS. The sample was separated on a C18 column (2.1×100 mm, 2.2 μm) and eluted with isocratic elution. Mass spectrometric detection was carried out on a thermo Q-exactive mass spectrometer with HESI. The scanning range of precursor ions was from m/z 50 to m/z 750. Result and Discussion: Through several indicators including standard curve, precision, accuracy, stability, matrix effect and recovery rate, the enzymatic kinetics parameters including T1/2, Vmax and CLint were completed. Several metabolites of TPL-MS were identified. Conclusion: UPLC-MS/MS method is an accurate and sensitive method for determination of TPL in liver microsome samples with good precision, accuracy and stability. The variation of parameters indicated that the microspheres can delay the elimination of TPL in liver microsomes. The metabolism of TPL-MS varied among species, but no new metabolites appeared.

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