Evidence for the involvement of a 35-kDa membrane protein in the synthesis of glucosylphosphoryldolichol

1990 ◽  
Vol 10 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Rick Drake ◽  
Grazyna Palamarczyk ◽  
Boyd Haley ◽  
William J. Lennarz
Keyword(s):  
Membrane Protein ◽  
Uv Light ◽  
Divalent Cations ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Sds Page ◽  
Molecular Masses ◽  
Weight Protein ◽  
35 Kda Protein ◽  
Page Electrophoresis

Photoactivatable (β-23P)5-azidoUDPGlc binds to two proteins in rat liver microsomes. As determined by SDS-PAGE electrophoresis, the molecular masses of the32P-labeled proteins were found to be 62-and 35-kDa. Binding of the photoprobe to both proteins was inhibited by addition of unlabeled UDPGlc. Labeling of the higher molecular weight protein occurred in the absence of photoactivation. In contrast, formation of the32P-labeled 35-kDa protein was dependent on exposure of the membranes to UV light (250nm). Moreover, labeling of the 35-kDa protein required the intact sugar nucleotide and divalent cations and was affected by the level of the endogenous and exogenous dolichylphosphate. All of these results are consistent with the possibility that the 35-kDa membrane protein is a component of glucosylphosphryldolichol synthase.

Lipid Peroxidation and Cherniluminescence during Naproxen Metabolism in Rat Liver Microsomes

1994 ◽  
Vol 13 (12) ◽  
pp. 831-838 ◽  
Author(s):  
Hiroyuki Yokoyama ◽  
Toshiharu Horie ◽  
Shoji Awazu
Keyword(s):  
Lipid Peroxidation ◽  
Singlet Oxygen ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Thiobarbituric Acid ◽  
Rat Liver Microsomes ◽  
Oxygen Species ◽  
Thiobarbituric Acid Reactive Substances ◽  
Microsomal Suspension ◽  
Weight Protein

1 Rat liver microsomal suspension containing NADPH and MgCl2 was incubated at 37°C with naproxen, a non-steroidal anti-inflammatory drug. Thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances were formed in the microsomal suspension. 2 Chemiluminescence was produced from the microsomal suspension. This chemiluminescence production was well correlated to the TBA-RS formation, indicating that the chemiluminescence production was closely associated with the lipid peroxidation. 3 The addition of SKF-525A to the microsomal suspension inhibited the production of TBA-RS, chemiluminescence and 6-demethylnaproxen (6-DMN), the oxidative product of naproxen. Further, the antioxidant, α-tocopherol and singlet oxygen quenchers like histidine, dimethylfuran and 1,4-diazabicyclo[2,2,2]octane strikingly inhibited the productions of chemiluminescence and TBA-RS. 4 Neither naproxen nor 6-DMN caused lipid peroxidation in the absence of NADPH. Thus, lipid peroxidation and chemiluminescence during the oxidation of naproxen in liver microsomes was suggested to be provoked by reactive oxygen species and an origin of chemiluminescence was shown to be singlet oxygen.

scholarly journals Studies on membrane proteins involved in ribosome binding on the rough endoplasmic reticulum. Ribophorins have no ribosome-binding activity

1987 ◽  
Vol 245 (3) ◽  
pp. 811-819 ◽  
Author(s):  
H Yoshida ◽  
N Tondokoro ◽  
Y Asano ◽  
K Mizusawa ◽  
R Yamagishi ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Rat Liver ◽  
Concanavalin A ◽  
Protein Fraction ◽  
Proteolytic Enzymes ◽  
Binding Capacity ◽  
Liver Microsomes ◽  
Binding Activity ◽  
Rat Liver Microsomes ◽  
Ribosome Binding

A membrane protein fraction showing affinity for ribosomes was isolated from rat liver microsomes (microsomal fractions) in association with ribosomes by treatment of the microsomes with Emulgen 913 and then solubilized from the ribosomes with sodium deoxycholate. This protein fraction was separated into two fractions, glycoproteins, including ribophorins I and II, and non-glycoproteins, virtually free from ribophorins I and II, on concanavalin A-Sepharose columns. The two fractions were each reconstituted into liposomes to determine their ribosome-binding activities. The specific binding activity of the non-glycoprotein fraction was approx. 2.3-fold higher than that of the glycoprotein fraction. The recovery of ribosome-binding capacity of the two fractions was about 85% of the total binding capacity of the material applied to a concanavalin A-Sepharose column, and about 90% of it was found in the non-glycoprotein fraction. The affinity constants of the ribosomes for the reconstituted liposomes were somewhat higher than those for stripped rough microsomes. The mode of ribosome binding to the reconstituted liposomes was very similar to that to the stripped rough microsomes, in its sensitivity to proteolytic enzymes and its strong inhibition by increasing KCl concentration. These results support the idea that ribosome binding to rat liver microsomes is not directly mediated by ribophorins I and II, but that another unidentified membrane protein(s) plays a role in ribosome binding.

scholarly journals Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato

1998 ◽  
Vol 334 (3) ◽  
pp. 571-576 ◽  
Author(s):  
A. Harvey MILLAR ◽  
Carina KNORPP ◽  
Christopher J. LEAVER ◽  
Steven A. HILL
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Divalent Cations ◽  
Pyruvate Dehydrogenase Complex ◽  
Protein Sequences ◽  
Protein Band ◽  
Sds Page ◽  
Molecular Masses ◽  
Α Subunits ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 µmol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were α subunits of pyruvate dehydrogenase, and the 37 kDa protein was the β subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.

scholarly journals ELECTRON MICROSCOPE EXAMINATION OF SUBCELLULAR FRACTIONS

1971 ◽  
Vol 51 (1) ◽  
pp. 52-71 ◽  
Author(s):  
Maurice Wibo ◽  
Alain Amar-Costesec ◽  
Jacques Berthet ◽  
Henri Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Membrane Protein ◽  
Distribution Pattern ◽  
Biochemical Analysis ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Microscope Examination ◽  
Membrane Area ◽  
Microsome Fraction

Rat liver microsomes and microsomal subfractions isolated by density equilibration were submitted to a quantitative morphological and biochemical analysis. The total area of the endoplasmic reticulum was estimated at 7.3 m2 per g of liver. The microsome fraction contained 2.8 mg of phospholipids and 6.7 mg of proteins per m2 of membrane area. After correction for ribosomal and intracisternal proteins, the latter value was lowered to 4.7 mg of membrane protein per m2. More than half of the microsomal vesicles carried ribosomes. After density equilibration of the microsomes, the distribution pattern of ribosomes followed closely that of RNA. The ribosome load of the microsomal vesicles increased steadily along the density gradient, indicating the existence of a continuous spectrum of microsomal entities ranging from entirely ribosome-free vesicles to vesicles heavily coated with ribosomes.

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.

scholarly journals Proteases from Canavalia ensiformis: Active and Thermostable Enzymes with Potential of Application in Biotechnology

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Rayane Natshe Gonçalves ◽  
Suellen Duarte Gozzini Barbosa ◽  
Raquel Elisa da Silva-López
Keyword(s):  
Divalent Cations ◽  
Peptidase Activity ◽  
Leaf Extracts ◽  
Thermostable Enzymes ◽  
Biotechnological Potential ◽  
Good Thermal Stability ◽  
Sds Page ◽  
Molecular Masses ◽  
Triton X ◽  
Different Levels

Extracts of leaves, seeds, roots, and stem from a tropical legume, C. ensiformis, were prepared employing buffers and detergent in aqueous solution. Leaf extracts had the highest protein content and the most pronounced peptidase activity with optimal pH in the neutral to alkaline range. All extracts exhibited peaks of activity at various pH values, suggesting the presence of distinctive classes of proteases. N-α-Tosyl-L-arginine methyl ester hydrolysis was maximal at 30°C to 60°C and peptidase activity from all extracts presented very good thermal stability after 24 h incubation at 70°C. C. ensiformis proteases exhibited molecular masses of about 200–57, 40–37, and 20–15 kDa by SDS-PAGE analysis. These enzymes cleaved hemoglobin, bovine serum albumin, casein, and gelatin at different levels. Serine and metalloproteases are the major proteases in C. ensiformis extracts, modulated by divalent cations, stable at 1% of surfactant Triton X-100 and at different concentrations of the reducing agent β-mercaptoethanol. Thus, C. ensiformis expresses a particular set of proteases in distinctive organs with high activity and stability, making this legume an important source of proteases with biotechnological potential.

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.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Nontargeted Metabolomics by High-Resolution Mass Spectrometry to Study the In Vitro Metabolism of a Dual Inverse Agonist of Estrogen-Related Receptors β and γ, DN203368

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 776
Author(s):  
Sin-Eun Kim ◽  
Seung-Bae Ji ◽  
Euihyeon Kim ◽  
Minseon Jeong ◽  
Jina Kim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Human Liver ◽  
High Resolution Mass Spectrometry ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Rat Liver Microsomes ◽  
High Resolution Mass ◽  
Resolution Mass

DN203368 ((E)-3-[1-(4-[4-isopropylpiperazine-1-yl]phenyl) 3-methyl-2-phenylbut-1-en-1-yl] phenol) is a 4-hydroxy tamoxifen analog that is a dual inverse agonist of estrogen-related receptor β/γ (ERRβ/γ). ERRγ is an orphan nuclear receptor that plays an important role in development and homeostasis and holds potential as a novel therapeutic target in metabolic diseases such as diabetes mellitus, obesity, and cancer. ERRβ is also one of the orphan nuclear receptors critical for many biological processes, such as development. We investigated the in vitro metabolism of DN203368 by conventional and metabolomic approaches using high-resolution mass spectrometry. The compound (100 μM) was incubated with rat and human liver microsomes in the presence of NADPH. In the metabolomic approach, the m/z value and retention time information obtained from the sample and heat-inactivated control group were statistically evaluated using principal component analysis and orthogonal partial least-squares discriminant analysis. Significant features responsible for group separation were then identified using tandem mass spectra. Seven metabolites of DN203368 were identified in rat liver microsomes and the metabolic pathways include hydroxylation (M1-3), N-oxidation (M4), N-deisopropylation (M5), N,N-dealkylation (M6), and oxidation and dehydrogenation (M7). Only five metabolites (M2, M3, and M5-M7) were detected in human liver microsomes. In the conventional approach using extracted ion monitoring for values of mass increase or decrease by known metabolic reactions, only five metabolites (M1-M5) were found in rat liver microsomes, whereas three metabolites (M2, M3, and M5) were found in human liver microsomes. This study revealed that nontargeted metabolomics combined with high-resolution mass spectrometry and multivariate analysis could be a more efficient tool for drug metabolite identification than the conventional approach. These results might also be useful for understanding the pharmacokinetics and metabolism of DN203368 in animals and humans.

scholarly journals The metabolism of gelsevirine in human, pig, goat and rat liver microsomes

2021 ◽  
Author(s):  
Hua‐Hai Zhang ◽  
Wen‐Jia Yang ◽  
Ya‐Jun Huang ◽  
Wen‐Jing Li ◽  
Shuo‐Xin Zhang ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Microsomes ◽  
Rat Liver Microsomes
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