Presence Of 2-mercaptoethanol Research Articles

Thermostable mannose-binding lectin fromDendrobium jindleyanumwith activities dependent on sulfhydryl content

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands byliquidchromatography-tandemmassspectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18,4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C 18 stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L −1 phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L −1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 μL s −1 a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 μL s −1 such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 μmol L −1 for Tyr to 0.51 μmol L −1 for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.

Interaction of β-lactoglobulin with ionic surfactants: Apparent molar volume and compressibility studies of ionic surfactants in aqueous solutions of β-lactoglobulin

In this work we present apparent molar volume ( Φ v), adiabatic compressibility ( Φ k) and sound velocity number [ U] of sodium dodecyl sulfate (SDS), sodium n-nonyl sulfate (SNS), sodium dodecyl benzene sulfonate (SDBS), cetyl trimethyl ammonium bromide (CTAB), and di (dodecyl dimethyl ammonium bromide) (12-2-12) in aqueous solution of β-lactoglobulin (βLG) between 10 and 36 °C at an interval of 2 °C. The temperature dependence of Φ v value of these surfactants is found to be completely consistent with temperature over the entire concentration range; two minima are seen to occur at 22 and 32 °C. The sharpness of the minima decreases with the increase in surfactant concentration. However, no such pattern is observed with Φ k value. Corresponding experiment with βLG (i.e., in absence of surfactant) shows two maximum at the same temperatures as does the minima in Φ v value of surfactants. [ U] and Φ k are found to contrast in their behaviour with regards to the effect of temperature and surfactant concentration. However, this is suggested to provide a consistent view as regards to the surfactant binding to protein. We fail to observe such a complex temperature dependence of Φ v value in the presence of 2-mercaptoethanol (a protein denaturant). Assuming complete denaturation of βLG, the surfactant binding to βLG is believed to be diversified into electrostatic and hydrophobic interactions.

Solid-phase synthesis of backbone-modified DNA analogs by the boranophosphotriester method using new protecting groups for nucleobases

Backbone-modified DNA analogs were synthesized in good yields by the boranophosphotriester method on a solid support. The oligodeoxyribonucleoside boranophosphates, protected with 2-(azidomethyl)benzoyl groups for nucleobases, were converted into DNA and its backbone-modified analogs via the corresponding H-phosphonate intermediates. A new protecting group for the O 6 position of 2′-deoxyguanosine, 4-azidobenzyl (ABn) group, was also developed. The ABn group can be quickly removed by treatment with MePPh 2 and H 2O in the presence of 2-mercaptoethanol.

Preparation and properties of transparent bulk polymer nanocomposites with high nanophase contents

Transparent bulk polymer nanocomposites with high contents of ZnS nanoparticles (NPs) were prepared by free radical initiated in-situ bulk polymerization of N,N-dimethylacrylamide (DMAA), styrene (St) and divinylbenzene (DVB) using 2,2′-azobisisobutyronitrile (AIBN) as initiator in the presence of 2-mercaptoethanol (ME) capped ZnS NPs. The structure and properties of the nanocomposites were studied. Sphalerite ZnS NPs with average size of about 3nm were dispersed homogeneously in polymer matrices. The ME capped ZnS nanophase reached 30 wt%, while the bulk nanocomposites still exhibited good transparency in the visible light range. TGA study showed that the nanocomposite materials had excellent thermal stability below 245 °C. Dynamic mechanical analysis and pencil hardness studies showed that the materials had good mechanical properties. However, with the increase of the ME capped ZnS content, the glass transition temperature of the nanocomposites decreased probably due to the plasticizing effect of the ME capped ZnS NPs. Refractive indices of the nanocomposite materials increased from 1.536 for the matrix to 1.584 upon increasing the weight fraction of the ME capped ZnS NPs to 30 wt%.

A principal component analysis and two-dimensional correlation infrared spectroscopy study on the thermal unfolding of ribonuclease A under reducing conditions

The analysis of protein unfolding pathways and the identification of specific conformational changes under perturbation are of fundamental importance for protein folding. In this study, thermally induced early unfolding events of bovine pancreatic ribonuclease A (RNase A) in the presence of 2-mercaptoethanol are evidenced successfully by principal component analysis and two-dimensional correlation infrared spectroscopy. The analyses of secondary structural changes in different stages have clearly distinguished the early events from the main unfolding in the temperature course of RNase A. It is revealed that in the early stage of the thermal unfolding subtle structural changes of the more temperature-sensitive β-sheet in RNase A dominates this process; while the main unfolding of RNase A is initiated with the structural changes of the more temperature-sensitive β-sheet, random coils and β-turns, followed by the less temperature-sensitive β-sheet component and the formation of a β-sheet-rich aggregate. The quantitative analysis of the power spectra reveals that the intensity variations from early stages are almost same in the scale, and each accounts for less than 1% that of the main unfolding. These findings demonstrate that the utilization of 2D IR spectrum and its power spectrum is powerful in exploring the minor early events and closely related structural changes of protein.

Purification, Characterization and N-terminal Sequences Alignment of a Mannose Specific Protein Purified from Potca Fish, Tetraodon patoca

A protein was isolated and purified from the ventral portion of the Potca fish, Tetraodon patoca. The method was accomplished by gel filtration of crude protein extract on Sephadex G-50 followed by Ion exchange chromatography on DEAE-cellulose and finally by affinity chromatography on ConA-Sepharose matrix. The molecular weight of the protein, determined by the gel filtration and SDS-PAGE was about 82,000 and 80,000 respectively, but 42,000 and 38,000 were indicated by SDS-PAGE in the presence of 2-mercaptoethanol. The protein agglutinated rat red blood cells and in a haptein-inhibition test, the protein was inhibited specifically by the D-mannose and mannose containing saccharides. The protein is glycoprotein with neutral sugar content of about 0.35%. The purified protein also showed strong cytotoxic effects, which was performed by brine shrimp lethality bioassay and histopathological examinations. The N-terminal amino acid sequences of both the subunits of the protein were also identified and used a blast search on N-terminal amino acid sequences of the subunits revealed that the protein showed significant homology with the homologous proteins in database.

EF24 Induces G2/M Arrest and Apoptosis in Cisplatin-resistant Human Ovarian Cancer Cells by Increasing PTEN Expression

We report that EF24, a synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one, greatly inhibits cisplatin-resistant (CR) human ovarian cancer cell proliferation. The inhibitory effect of EF24 on cell proliferation is associated with G(2)/M phase cell cycle arrest and increased G(2)/M checkpoint protein (pp53, p53, and p21) levels. Within 24 h following treatment, EF24 induced apoptosis in CR cells. The apoptosis was partially blocked by the general caspase inhibitor z-VAD. Within 12 h, EF24 induced a membranous FasL expression, consistent with a substantial decrease in the Ser(473) and Thr(308) phosphorylation of Akt, a known negative regulator of FasL transcription. Also, EF24 activated the phosphorylated PTEN and marginally up-regulated total PTEN expression through the inhibition of ubiquitin-mediated PTEN degradation. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt phosphorylation at Ser(473) and Thr(308), resulting in decreased apoptosis and increased cell survival. On the other hand, overexpression of PTEN markedly induced apoptosis. Our results clearly suggested that EF24 induced significant increase in PTEN expression. The up-regulation of PTEN inhibited Akt and MDM2, which enhanced the level of p53, thereby inducing G(2)/M arrest and apoptosis. Therefore, EF24 appears to have a potential therapeutic role in human ovarian cancer through the activation of PTEN.

Controllable synthesis of functionalized CdS nanocrystals and CdS/PMMA nanocomposite hybrids

We reported controllable synthesis of CdS nanocrystal-polymer transparent hybrids by using polymethylmethacrylate (PMMA) as a polymer matrix. In a typical run, the appropriate amounts of cadmium chloride (CdCl 2) and sodium sulfide (Na 2S) in the presence of 2-mercaptoethanol (ME) as the organic ligand are well dispersed in H 2O/DMF solution without any aggregation. From a combination of transmission electron microscopy (TEM) and a computing method of Brus’s model according UV–vis absorption spectra, the particle size of as-prepared hydroxyl-coated CdS nanocrystal was determined to be about 5 nm. Then, with the surface treatment with methacryloxypropyltrimethoxysilane (MPS), CdS–PMMA hybrids were obtained via free radical polymerization in situ. FT-IR characterization indicates the formation of robust bonding between CdS nanocrystals and the organic ligand and the formation of double-bond functional CdS nanocrystals. The TGA measurement displays CdS–PMMA hybrids possess better thermal stability compared with pure PMMA polymer. The fluorescence measurement shows that CdS nanocrystals and CdS–PMMA hybrids exhibit good optical properties. Also, the luminescent photographs taken under ultraviolet light prove the luminescence properties.

Controllable synthesis of ZnS/PMMA nanocomposite hybrids generated from functionalized ZnS quantum dots nanocrystals

We reported controllable synthesis of ZnS nanocrystal-polymer transparent hybrids by using polymethylmethacrylate (PMMA) as a polymer matrix. In a typical run, the appropriate amounts of zinc chloride (ZnCl2) and sodium sulfide (Na2S) in the presence of 2-mercaptoethanol (ME) as the organic ligand were well dispersed in H2O/dimethylformamide solution without any aggregation. In addition, the Mn-doped ZnS nanocrystals (NCs) were synthesized with similar method. Then, ZnS-PMMA hybrids were obtained via free radical polymerization in situ by using ZnS NCs functionalized with methacryloxypropyltrimethoxysilane (MPS). FT-IR characterization indicates the formation of robust bonding between ZnS NCs and the organic ligand. The TEM images show that ZnS NCs are well dispersed in PMMA matrix, and particle size of as-prepared ZnS NCs is about 2.6 nm, in agreement with the computing results of Brus’s model and Debye–Scherrer formula. The photoluminescence measurements present that ZnS NCs, Mn-doped ZnS NCs, and ZnS/PMMA hybrid show good optical properties.

Electrophoretic and immunochemical techniques for the molecular reassessment of relationships within Vicieae

In this study, an array of electrophoretic and immunochemical techniques was used to investigate the legumins, vicilins and albumins of seed storage proteins inPisum sativum,Vicia faba,Lens esculentum, andCicer arietinumto delimit the boundary of the tribeVicieaeand to clarify the systematic position of the genusCicer. The band patterns of the legumins of these species were broadly similar in that they had bands at Mr 60 kDa which disappeared in the presence of 2-mercaptoethanol, giving rise to two sets of new bands, at Mr approximately 40 kDa and 20 kDa, representing acidic or α and basic or β subunits. The band patterns of the vicilins were also quite similar in that they showed bands at Mr approximately 71 kDa (convicilin) and 50 kDa (vicilin), which were not altered by the presence of 2-mercaptoethanol. Serologically, the legumins ofVicia fabaandLens esculentumexhibited total identity withPisumlegumin antiserum under nonreducing conditions, whereas the legumin ofCicer arietinumexhibited only partial identity, which was attributed to the failure of the low molecular subunit pair (Mr 33 kDa) to react withPisumlegumin antiserum. On the other hand, the vicilins ofVicia faba,Lens esculentumandCicer arietinumhad only partial identity with the vicilin ofPisum sativum, which was due to the failure of a number of subunits along the electrophoretic patterns of these species to react withPisum sativumvicilin antiserum. The electrophoretic patterns ofVicia faba,Lens esculentumandCicer arietinumwere markedly different for the albumins. However, immunochemically they gave a positive reaction withPisummajor albumin antiserum (Mr 25 kDa) and showed a band with a molecular weight slightly higher than the major albumin ofPisum sativum. Extending the immunochemical study to members of thePhaseoleae, Glycineae, CajaneaeandDiocleaerevealed that the vicilin and legumin ofCicerwere more closely related to theVicieaethan to these tribes. Thus the data presented in this work recommended the classification ofCicerunderVicieaerather than as a separate tribeCicerideae.

Cloning and expression of the gene of the human chymotrypsin-like kallikrein-7 protease in Escherichia coli and isolation of the recombinant protein

Amplification of a segment coding for the synthesis of the chymotrypsin-like kallikrein-7 protease (KLK-7) and its cloning into the expression plasmid pET23a (+) were performed on a sample of human cDNA. The biosynthesis of KLK-7 in transformed E. coli BL21(DE3) cells was accompanied by the formation of insoluble inclusion bodies. Recombinant KLK-7 was extracted from the inclusion bodies with 7 M urea in the presence of 2-mercaptoethanol, purified by methods of metal-chelate and ion exchange chromatography, converted to a soluble form, and used for the production of a monospecific antiserum.

Reactions of Sulfenic Acid with 2-Mercaptoethanol: A Mechanism for the Inhibition of Gastric (H+−K+)-Adenosine Triphosphate by Omeprazole

The reactions of omeprazole, a potent proton pump inhibitor (PPI) were investigated in the presence of 2-mercapotoethanol. Reactions were monitored in solutions buffered to pH values ranging 2.0-8.0 using differential pulse polarography (DPP) at the static mercury drop electrode (SMDE). The fast, sensitive and selective electrochemical technique facilitated successive recordings of voltammograms (peak current (nA) vs. peak potential (volts vs. Ag/AgCl)) for all analytes in situe, including the 2-mercaptoethanol. In acidic solutions and in the presence of 2-mercaptoethanol, omeprazole undergoes degradation into three compounds, the first is a cyclic sulfenamide (D+), previously believed to be the active inhibitor of the H+, K+-ATPase, the second is the omeprazole dimer, and the third is the disulfide believed to be the product of reaction between 2-mercaptoethanol and D+. The cyclic sulfenamide (D+) solution was found to be stable in solutions containing 2-mercaptoethanol having pH values: 2.0, 4.0, and 6.0. This finding proved conclusively that the cyclic sulfenamide is not reactive toward the 2-mercaptoethanol. In contrast to previous reports, the conversion of the sulfenic acid intermediate into D+ was found to be irreversible. Due to this irreversibility, D+ and sulfenic acid were not rapidly interconvertable. The present work suggests that the active inhibitor is the sulfenic acid.

The first dinuclear copper(ii) and zinc(ii) complexes containing novel Bis-TACN: syntheses, structures, and DNA cleavage activities

Two novel binuclear complexes [Cu(2)(L)].(ClO(4))(2) (1) and [Zn(2)(L)].(ClO(4))(2) (2) were synthesized and crystallographically characterized {L = 1(4),5(4)-dimethyl-1(2),5(2)-dihydroxy-1(1,3),5(1,3)-dibenzene-3(1,4),7(1,4)-di-1,4,7-triazacyclononane}. The cation [Cu(2)(L)](2+) structure of 1 is similar to that of [Zn(2)(L)](2+) of 2. The central ion is bridged by the di-phenoxo of L and lies in a close to perfect square pyramidal geometry. 1 and 2 crystallize in the triclinic space group P1. The two complexes effectively promote the cleavage of plasmid DNA in the presence of activating agents at physiological pH and temperature. The pseudo-Michaelis-Menten kinetic parameters k(cat) = 1.61 h(-1), K(m) = 1.35 x 10(-5) M for complex 1 in the presence of mercaptoethanol; k(cat) = 2.48 h(-1), K(m) = 5.5 x 10(-5)M for complex 2 in the presence of hydrogen peroxide were obtained. The mechanism of plasmid DNA cleavage was studied by adding standard radical scavengers. DNA cleavage reaction by the binuclear Zn(II)/H(2)O(2) system is a hydrolytic mechanism.

Synthesis of Nanocrystal−Polymer Transparent Hybrids via Polyurethane Matrix Grafted onto Functionalized CdS Nanocrystals

We reported the first synthesis of CdS nanocrystal-polymer transparent hybrids by using polyurethane (PU) grafted onto quantum dots (QDs) CdS nanocrystals. In a typical run, the appropriate amounts of cadmium chloride (CdCl2) and sodium sulfide (Na2S) in the presence of 2-mercaptoethanol (ME) as the organic ligand are well dispersed in H2O/DMF solution without any aggregation. From a combination of transmission electron microscopy (TEM) and a computing method of Brus's model according to UV-vis absorption spectra, the particle size of as-prepared hydroxyl-coated CdS nanocrystals is about 5 nm. Then, PU-CdS transparent nanocomposites hybrids were synthesized by a two-step reaction. The effect of the different ratios of ME/Cd2+ and H2O/DMF on the resulting particle size of CdS nanocrystals was investigated by UV-vis absorption measurements. FT-IR and TGA characterizations indicate the formation of robust bonding between CdS nanocrystals and the organic ligand. The fluorescence measurement shows that CdS-PU hybrids exhibit good optical properties.

Changes in vitreous amino acid concentrations in a rabbit model of cataract surgery

We previously reported marked elevation of glutamate, gamma-aminobutyric acid (GABA) and alanine in the vitreous after combined cataract surgery and vitrectomy for macular disorder. Photo-stimulation by the operating microscope and increased intraocular pressure (IOP) are possible causes of postoperative increases in amino acids. An animal model was used to verify this hypothesis. Forty-five coloured rabbits were studied; stress was applied to simulate cataract surgery. Simultaneous photic and pressure stress, photic stress alone, or pressure stress alone were exerted for 20 mins. The vitreous was extracted at the end of the stress period, or 10 mins or 30 mins later. Levels of six amino acids (asparate, glutamate, glycine, taurine, alanine and GABA) in the vitreous were analysed quantitatively. Significant increases in vitreous glutamate concentrations in treated eyes were observed following simultaneous photic and pressure stress, as well as after pressure stress alone, whereas no difference was found after photic stress alone. The mean glutamate concentrations in treated eyes and fellow eyes, respectively, were 5.59 +/- 2.03 microM and 4.36 +/- 2.09 microM (p < or = 0.05) 10 mins after simultaneous photic and pressure stress, and 4.32 +/- 0.97 microM and 2.29 +/- 0.51 microM (p < 0.05) 10 mins after pressure stress alone. Taurine concentration was elevated by pressure stress alone at 0 min and at 10 mins post-stress. Alanine concentration was reduced by photic stress alone at 10 mins post-stress. In all protocols, no significant differences between treated and fellow eyes were observed at 30 mins after stress. Time-dependent changes were observed not only in the treated eyes, but also in untreated fellow eyes. Elevations of vitreous glutamate in cataract surgery may be caused by pressure stress due to irrigation. Photic stress caused by the operating microscope has little influence. Sympathetic response may be involved in amino acid changes due to stress in cataract surgery.

The Iron-Sulfur Cluster-free Hydrogenase (Hmd) Is a Metalloenzyme with a Novel Iron Binding Motif

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing cofactor of yet unknown structure. X-ray absorption spectroscopy of the active, as isolated enzyme from Methanothermobacter marburgensis (mHmd) and of the active, reconstituted enzyme from Methanocaldococcus jannaschii (jHmd) revealed the presence of mononuclear iron with two CO, one sulfur and one or two N/O in coordination distance. In jHmd, the single sulfur ligand is most probably provided by Cys176, as deduced from a comparison of the activity and of the x-ray absorption and Mössbauer spectra of the enzyme mutated in any of the three conserved cysteines. In the isolated Hmd cofactor, two CO, one sulfur, and two nitrogen/oxygen atoms coordinate the iron, the sulfur ligand being most probably provided by mercaptoethanol, which is absolutely required for the extraction of the iron-containing cofactor from the holoenzyme and for the stabilization of the extracted cofactor. In active mHmd holoenzyme, the number of iron ligands increased by one when one of the Hmd inhibitors (CO or KCN) were present, indicating that in active Hmd, the iron contains an open coordination site, which is proposed to be the site of H2 interaction.

Serum high-density and low-density lipoprotein cholesterol determined by ultracentrifugation and high-performance liquid chromatography

目的 建立一种新的预期用作参考方法的高密度脂蛋白胆固醇(HDLC)和低密度脂蛋白胆固醇(LDLC)准确测定方法.方法 血清与含2-巯基乙醇(ME)和脯氨酸的溴化钠溶液混合,使背景密度为1.063,超速离心分离高密度脂蛋白(HDL);血清与含脯氨酸的溴化钠溶液混合,使背景密度为1.006,超速离心分离HDL和低密度脂蛋白(LDL);用高效液相色谱测定两种脂蛋白组分胆固醇.结果 ME和脯氨酸可有效消除脂蛋白(a)对超速离心法分离HDL的影响;新方法测定HDLC和LDLC的总变异系数分别为0.96%～2.07%和0.65%～1.12%.结论 建立血清HDLC和LDLC准确测定方法,方法可靠、精密、简便,可望用作HDLC和LDLC测定参考方法。

Purification and characterization of two endoglucanases from Melanocarpus sp. MTCC 3922

This study reports the purification and characterization of endoglucanases (EG I and EG II) from a newly isolated thermophilic fungus, Melanocarpus sp. MTCC 3922. The molecular weight of EG I and EG II as with SDS-PAGE and p I were ∼40 and 50 kDa, and ∼4.0 and 3.6, respectively. EG I and EG II were optimally active at 50 and 70 °C, and pH 6.0 and 5.0, respectively. EG I was active over a broad range of pH (5.0–7.0), whereas, loss of activity was observed as the temperature was increased from 50 to 80 °C. However, EG II was active over pH 4.0–6.0 and temperature 40–80 °C. The presence of mercaptoethanol and SDS inhibited the EG I activity but showed no negative effect on EG II. Both the endoglucanases showed higher activity against barley-β-glucan as compared to CMC. K m values of EG I and EG II for barley-β-glucan were lower than CMC. Turn over number ( K cat) and catalytic efficiency ( K cat/ K m) values of both the endoglucanases were higher with barley-β-glucan as substrate than CMC. EG I showed affinity for Avicel indicating the presence of cellulose binding domains (CBD) whereas, EG II was found to lack CBD.

Formation of Retinoylated Proteins from Retinoyl-CoA in Rat Tissues

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.