Simultaneous analysis of esterase and transferase activities in cytosol proteins from the bovine retina by using microscale non-denaturing two-dimensional electrophoresis

Esterase and transferase activities were analyzed simultaneously after cytosol proteins in the bovine retina were separated by microscale non-denaturing two-dimensional electrophoresis (2-DE). Esterase activity was specifically inhibited by an esterase inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), and transferase activity was specifically inhibited by a glutathione S-transferase (GST) inhibitor, 2-phenyl-1,2-benziso selenazol-3(2H)-one (ebselen). Both esterase and transferase were precipitated when ammonium sulfate was added to the cytosol up to 50% saturation (50% AS fraction), and were detected in the 50% AS fraction by using the 2-DE. After the cytosol proteins in the 50% AS fraction were separated by using non-denaturing 2-DE, polypeptides of the separated proteins were identified by peptide mass fingerprinting and post-source decay analysis by using MALDI-MS, or by immunoreactivity by using a specific antibody. The spots of esterase and transferase activities in the 2-DE pattern were identified as phosphodiesterase and GST, respectively. This simultaneous analysis of enzyme activities can be applied to screen-specific or non-specific medicines which affect enzyme activities.     

Analysis of hydrolytic activity of phospholipase Calpha from porcine retina on retinyl ester and phosphatidylcholine using non-denaturing two-dimensional electrophoresis and mass spectrometry

Hydrolysis of retinyl esters and phospholipids is important for visual functions of the animal retina. This study aimed to examine hydrolytic activity of an enzyme with native substrates such as retinyl esters and phospholipids responsible for this function in porcine retina. After cytosolic proteins were extracted from porcine retina, the proteins were separated using non-denaturing two-dimensional electrophoresis (2DE). Some major proteins and phospholipase Calpha were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) or electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). The phospholipase Calpha showed hydrolytic activities with not only alpha-naphtyl acetate but also with retinyl palmitate and phosphatidylcholine when effects of different substrates were investigated using enzyme activity staining on 2DE or MALDI-TOF-MS. Results indicated that hydrolytic activity of the enzyme with non-native and native substrates could be examined using a combination of non-denaturing 2DE and MALDI-TOF-MS.     

Hydrolytic activity of lipase on anion-exchange solid phase column after separation and electrotransfer by non-denaturing electrophoresis

This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.     

Hydrolytic activity of lipase on anion-exchange solid phase column after separation and electrotransfer by non-denaturing electrophoresis

This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using α-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell™ Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.     

Production of enzyme reactors after separation by non-denaturing two-dimensional electrophoresis and immobilization on membrane

Activities of carboxylesterase and malate dehydrogenase on membranes were retained after enzymes of mouse liver cytosol were separated by non-denaturing, two-dimensional electrophoresis (2-DE), stained using imidazole and zinc salts and electroblotted on to membranes. Furthermore, hydrolytic changes of phosphatidylcholine by the esterase were examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after separation, and reversible staining and immobilization to membranes. Hydrolytic activity of the esterase on the membranes was 20% of the original activity of the tissue homogenate. The present method can be applied to the production of several types of enzyme reactors on membranes.     

Protein spot recognition on the non-denaturing and denaturing two-dimensional electrophoresis patterns using in situ immunosubtraction via Protein A agarose and antibodies.

Specific proteins in small amounts of human plasma were subtracted from patterns of non-denaturing two-dimensional electrophoresis (non-denaturing 2-DE) by layering a 7 microl aliquot of Protein A agarose-antibody complex on the top of an isoelectric focusing (IEF) gel before the loading of a plasma sample. The Protein A agarose suspension was recovered after non-denaturing IEF and was mixed with 8 M urea-5% 2-mercaptoethanol-1% NP-40 to extract the antibody and the specific plasma protein from Protein A agarose. The extract was then subjected to denaturing two-dimensional electrophoresis (denaturing 2-DE) and the location of the specific polypeptide was determined. The technique can be applied to the extraction and analysis of proteins in small amounts of samples.     

Heterogeneity of the glucocorticoid receptors: molecular transformations during activation, detected by electrofocusing

Isoelectric focusing (IEF) of glucocorticoid receptor (GR) of the neural retina of the 14-day chick embryo was conducted under conditions that yielded quantitative recovery of binding activity. IEF of the cytosol, equilibrated with [3H]triamcinolone acetonide (TA) at 0-2 degrees C yielded three major TA-GR components with apparent isoelectric points (pI') of 5.4 +/- 0.3, 6.5 +/- 0.2, and 7.6 +/- 0.3, designated as I, II, and III, respectively. During temperature-induced activation (incubation at 30 degrees C for 60 min, in the presence of free [3H]TA and 0.15 M KCl), approximately 25% of the specifically bound TA was irreversibly lost. IEF reveals that this loss is accounted for by the complete loss of binding from I. During activation, II also decreases but correspondingly III increases, i.e., the sum of II and III remains unchanged. Only the bound TA of I is sensitive to the addition of KCl (a promoter of activation). This sensitivity of I is temperature dependent. Molybdate (an inhibitor of activation) protects the bound TA of I and suppresses the formation of III. These two effects of molybdate diminish simultaneously when the temperature is increased to 30 degrees C. III preferentially exhibits binding activity to nuclei. The data suggest that (i) the glucocorticoid-free cytosol contains two GRs, I and II, with possibly two different functions; (ii) activation involves the loss of bound TA from I and the transformation of II to III with increased pI; (iii) these two molecular events in GR activation are interdependent.     

Isoelectric focusing and 2D electrophoresis of the human androgen receptor.

Nuclear androgen receptors from cultured genital skin fibroblasts were analyzed by non-denaturing isoelectric focusing (IEF) in ultrathin polyacrylamide gels before and after photoaffinity labeling with [3H]methyltrienolone. Both reversibly and covalently labeled receptors focused at pH 5.28 +/- 0.20 when extracted from nuclei with high salt. Lowering of the salt concentration yielded, in both cases, a second species which focused at pH 7.16. This species became predominant when nuclei were sonicated in IEF sample buffer containing no salt, even after extensive nucleic acid digestion. Low salt cytosols from both prostate and foreskin focused as a single peak of pI: 4.93 +/- 0.31 which remained unchanged when KCl was added to the cytosol up to a concentration of 0.6 M. SDS-polyacrylamide gel electrophoresis of photoaffinity labeled receptors revealed labeled proteins with Mw 90-95 kDa. Two-dimensional electrophoresis of photoaffinity labeled nuclear receptors, extracted in low or high salt, showed that the two isoforms (pI 5.28 and 7.16) contain the same steroid-binding subunit with Mw 90-95 kDa. Nuclear receptors from 4 patients with the receptor positive form of the Complete Androgen Insensitivity Syndrome (CAIS, Rc+) were analyzed by non-denaturing IEF: a single species was observed, focusing at pH 6.0 whether in high or low salt conditions. These results indicate that the nuclear androgen receptor is an acidic protein with pI 5.28 and Mw 90-95 kDa under maximum protein dissociation conditions. When extracted under low salt conditions, it can be isolated in a neutral form (pI 7.16) suggesting its association with a nuclear protein. Receptors of (CAIS, Rc+) patients have an abnormal charge and show no pI shift upon lowering of the salt concentration suggesting that this shift could be a significant step in the mechanism of action of androgens.     

Multimodal analysis of metals in copper–zinc superoxide dismutase isoforms separated on electrophoresis gels

It has been suggested that copper–zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.     

Direct analysis of retinal dehydrogenase activity on an electroblotting membrane following separation by non-denaturing two-dimensional electrophoresis

The reaction from retinal to retinoic acid catalyzed by retinal dehydrogenase on a polyvinylidene difluoride (PVDF) membrane was examined using laser desorption ionization time of flight mass spectrometry (LDI-TOF MS) when the enzyme was separated by non-denaturing two-dimensional electrophoresis (2-DE), transferred onto the membrane, and stained without impairing the enzyme activity. Furthermore, the enzyme was analyzed by de novo sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after proteins from mouse liver were separated by non-denaturing 2-DE, blotted onto the membrane, and stained. The results indicated that the reported methods could be applied for the direct examination of changes in retinoid catalyzed by enzymes on such membranes.     

Vitamin A receptors. II. Characteristics of retinol binding in chick retina and pigment epithelium

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [3H]retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [3H]retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of KD about 1 - 10(-9) and 4 - 10(-8).     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition

The mutagenicity of quercetin towards Salmonella typhimurium TA98 was enhanced by rat-liver cytosol. The enhancing factors in the cytosol were separated into 4 fractions by gel filtration, and one of them was identified as superoxide dismutase (SOD). It is suggested that SOD increases the mutagenicity of quercetin by protecting it from degradation.     

Effects of melatonin and light on porphyrin synthesis in the bovine retina, pigment epithelium and choroid.

The quantity of porphyrin synthesized in the presence of 10(-3) M delta-aminolevulinic acid (ALA) is several times higher in the bovine pigment epithelium than in the retina. Synthesis in the retina was found to be increased by illumination, whereas synthesis in the pigment epithelium was decreased if the whole anatomical unit (retina-pigment epithelium-choroid) was cultivated together. The quantity of porphyrin synthesized in the presence of 10(-3) M ALA or 10(-6) M melatonin was different when the pigment epithelium and retina were separated. The combination 10(-3) M ALA with 10(-6) M melatonin inhibited retinal porphyrin synthesis after green light adaptation, while in the pigment epithelium green light adaptation induced porphyrin synthesis. It is postulated that the light-sensitive porphyrin-haeme synthesis of the retina-pigment epithelium-choroid functional unit may serve and modulate the synthesis of guanylate cyclase for cGMP.     

Vitamin A receptors. I. Comparison of retinol binding to serum retinol-binding protein and to tissue receptors in chick retina and pigment epithelium.

1. A simple, efficient three-step method for purification of serum retinol-binding-protein is described with homogeneity obtained after chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. 2. Evidence is presented indicating that retinol receptors present in the cytosol fraction of chick retina and pigment epithelium are separate and distinct from purified retinol-binding protein. Fluorescence characteristics are different in tissue cytosol and serum as assessed by sucrose density gradient analysis. Tissue retinol receptors do not interact with human serum prealbumin although the prealbumin readily complexes with purified chicken retinol-binding protein. Likewise, no binding to serum retinol-binding protein antibody could be detected by sucrose density gradient analysis, in immunoprecipitation experiments or by double immunodiffusion. It thus appears that specific retinol receptors are present in neural retina and pigment epithelium that are different from serum retinol-binding protein.     

Vitamin A receptors: II. Characteristics of retinol binding in chick retina and pigment epithelium

Gel filtration studies demonstrate that retinol receptors of chick retinal and pigment epithelial cytosols are (1) of very similar nature (2) of small molecular size (about 18 000 daltons) and are different in character from serum proteins. Citral inhibits the binding of [³H] retinol to the retinal 2 S receptor. Retinol acetate competes with retinol for binding to 2 S receptor in both retina and pigment epithelium whereas retinol palmitate is an effective competitor only in the pigment epithelium. Dithiothreitol maximizes 2 S binding in retina and pigment epithelial cytosol; its absence does not lead to receptor aggregation however. A limited number of high affinity binding sites (2 S receptor) appear to be present in retina and pigment epithelium. A 5 S binding species is also present in pigment epithelium; it is similar in character to [³H] retinol binding in serum and may arise from serum contamination of the pigment epithelial preparation. Binding affinity in retina is high with possibly two classes of retinol binding sites present of K_D about 1·10^?9 and 4·10^?8.     

Increased Superoxide Dismutase Activity in Aged Human Cerebrospinal Fluid and Rat Brain Determined by Electron Spin Resonance Spectrometry Using the Spin Trap Method

Superoxide dismutase (SOD) activity in CSF of patients was determined by electron spin resonance spectrometry using the spin trap method. Variation in SOD activity was found among patients. SOD activity in CSF of subjects increased with age and this was identified as Cu,Zn-SOD activity by electrophoresis. In addition, animal experiments showed that SOD activities were higher in mitochondrial and cytosol fractions of aged rats than in those of adult rats. This finding on aged rat brain validates the increase of SOD activity in aged human CSF.     

Radioautographic study of the cellular proliferation of retinal pigment epithelium in axolotls

The proliferative activity of the pigment epithelium cells in the axolotl eyes was studied using 3H-thymidine in two types experiments: after the removal of lens, iris and retina and upon the cultivation of the pigment epithelium pieces in the cavity of lens-less eye. Irrespective of the operation type, the level of proliferation of the pigment epithelium cells changed regularly with respect to the time of observation. In the intact eye, the level of proliferation of the pigment epithelium cells was not high: the index of labelled nuclei equaled 0.5%, no mitoses were found. The highest values of the index of labelled nuclei (12.6-32.1%) and of the mitotic index (0.54-1.07%) were registered on the 10-20th days after the operation. After 40 days, the indices of proliferative activity of the pigment epithelium cells approached gradually those for the intact eye. The cultivation of the pigment epithelium cells in the cavity of a lens-less eye for 50 days did not result in their transdifferentiation into retina cells. The layered retina found in 7.7% of cases after the removal of lens, iris and retina could regenerate either from the cells of the retina growth zone localized in the region of embryonic split, or due to transdifferentiation of the pigment epithelium cells.     

Light-Induced Dopamine Release from Teleost Retinas Acts as a Light-Adaptive Signal to the Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules migrate in and out of the cells' long apical projections in response to changes in light condition. When the RPE is in its normal association with the retina, light onset induces pigment granules to disperse into the apical projections; dark onset induces pigment granules to aggregate into the cell bodies. However, when the RPE is separated from the retina, pigment granule movement in the isolated RPE is insensitive to light onset. It thus seems likely that a signal from the retina communicates light onset to the RPE to initiate pigment dispersion. We have examined the nature of this retina-to-RPE signal in green sunfish, Lepomis cyanellus. In isolated retinas with adherent RPE, light-induced pigment dispersion in the RPE is blocked by treatments known to block Ca2+-dependent transmitter release in the retina. In addition, the medium obtained from incubating previously dark-adapted retinas in the light induces light-adaptive pigment dispersion when added to isolated RPE. In contrast, the medium obtained from incubating dark-adapted retinas in constant darkness does not affect pigment distribution when added to isolated RPE. These results are consistent with the idea that RPE pigment dispersion is triggered by a substance that diffuses from the retina at light onset. The capacity of the conditioned medium from light-incubated retinas to induce pigment dispersion in isolated RPE is inhibited by a D2 dopamine antagonist, but not by D1 or alpha-adrenergic antagonists. Light-induced pigment dispersion in whole RPE-retinas is also blocked by a D2 dopamine antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of activity and mass spectrometric identification of mouse liver carboxylesterase and aldehyde dehydrogenase separated by non-denaturing two-dimensional electrophoresis after extraction with detergents

To examine the activities and identity of enzymes associated with organelles such as microsomes and mitochondria, proteins from mouse liver were extracted using the non-ionic detergents Nonidet P-40 (NP-40), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene isooctylphenyl ester (Triton X), n-octyl beta-D-glucoside (octyl glycoside) or anionic detergent sodium dodecylsulfate (SDS) after the removal of cytosolic proteins. The proteins extracted by detergents were separated by non-denaturing two-dimensional electrophoresis (2-DE). The activities of esterase and aldehyde dehydrogenase were retained by non-denaturing 2-DE after treatment with each non-ionic detergent, but the activities were reduced or lost when the proteins were extracted with more than 0.5% SDS. For proteomic analysis of the organelle-associated proteins in mouse liver, proteins were separated by non-denaturing 2-DE and were identified using electrospray ionization tandem mass spectrometry (ESI-MS/MS) after the proteins were solubilized by octyl glycoside, NP-40 and 0.1% SDS. Several organelle-associated proteins such as carboxylesterase, aldehyde dehydrogenase, glucose regulated protein and HSP60 were identified. These results indicate that the activities and identity of detergent-soluble enzymes can be examined by this non-denaturing 2-DE and mass spectrometry.