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.     

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.     

Simple quantification of Cu,Zn-superoxide dismutase activity after separation by non-denaturing isoelectric focusing

The Cu,Zn-superoxide dismutase (SOD) activity in bovine retina cytosol was separated from retinal pigment using short-length non-denaturing isoelectric focusing (IEF) (15-mm long x 1.3-mm i.d. column) and detected using non-denaturing two-dimensional electrophoresis (2-DE). After the SOD and pigment in the retina cytosol are separated, SOD activity can be quantified by water-soluble tetrazolium salt. We also found that SOD separated by this IEF retained its native function.     

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.     

Protein degradation by the phosphoinositide-specific phospholipase C-alpha family from rat liver endoplasmic reticulum.

A 60-kDa protein homologous to phosphoinositide-specific phospholipase C-alpha was purified to apparent homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the rough endoplasmic reticulum of rat liver through three sequential chromatographies on DEAE Toyopearl 650, AF-heparin Toyopearl 650M, and TSK gel G3000SW. The purified protein was monomeric, with an M(r) of 60,000. Eight types of protein were further separated from the 60-kDa protein and named ER60A-ER60H according to the order of their elution from a TSK gel DEAE-5PW column. They were essentially identical in terms of immunochemical properties and the NH2-terminal amino acid sequence. The partial amino acid sequence of ER60F showed homology to that of phosphoinositide-specific phospholipase C-alpha. ER60A-ER60H showed no phosphoinositide-specific phospholipase C activity. However, ER60A-ER60H catalyzed cleavage of themselves and the endoplasmic reticulum proteins protein disulfide-isomerase and calreticulin. Proteolytic degradation was inhibited by p-chloromercuribenzoate. These results indicate that ER60A-ER60H comprise a group of endoplasmic reticulum resident proteins and show thiol group-related proteolytic activity.     

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.     

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.     

Purification and activity of two phospholipase enzymes from Naja nigricolis nigricolis Reinhardt venom

Two phospholipase enzymes NN1 and NN2 were purified from the venom of Naja nigricolis nigricolis Reinhardt to apparent homogeneity. NN1 was purified by a two-step anion-exchange chromatography on DEAE-cellulose column while NN2 was purified by a combination of anion-exchange chromatography and gel filtration on Sephadex G-150. The enzyme NN1 moved homogenously on acrylamide gel as a monomer with a molecular weight of 65 kDa while NN2 was a dimer of 71 kDa. Both enzymes were clearly separated. Both enzymes hydrolyzed L-alpha-phosphatidyl choline with activities of 345.5 for NN1 and 727.8 micromol min(-1) x mg(-1) for NN2. The dimeric 71-kDa enzyme has a higher haemolytic and anticoagulant activity than the monomeric 65-kDa enzyme. It is apparent that the dimeric enzyme has a more pronounced activity than the monomer has, thus toxic activity may be related to the hydrolysis of phospholipids.     

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.     

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.     

Simultaneous production of immunoaffinity membranes

We simultaneously separated antibodies for transferrin, the third component of complement (C3), haptoglobin and transthyretin by multi-sample non-denaturing two-dimensional electrophoresis (2-DE), transferred them to a polyvinylidene difluoride (PVDF) membrane and then stained them using direct blue 71 to obtain membrane-immobilized antibodies. The antigens, transferrin, C3, haptoglobin and transthyretin were specifically bound to the membrane-immobilized antibodies and were eluted only after rinsing the membrane with acid solution. The antigens specifically bound to the membrane-immobilized antibodies were separated by SDS-PAGE and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, transferrin and transthyretin were trapped and eluted by each membrane-immobilized antibody and detected by MALDI-TOF MS directly without separations. Using membrane-immobilized anti-transferrin antibody, transferrin in flowing blood was directly trapped and analyzed. The results indicated that membrane-immobilized antibodies are simultaneously produced, and that the immunoaffinity membranes can capture specific substances in flowing fluids.     

Production,purification and characterization of an extracellular lipase from Aureobasidium pullulans HN2.3 with potential application for the hydrolysis of edible oils

Lipase production (8.02 ± 0.24 U/ml) by the yeast Aureobasidium pullulans HN2.3 isolated from sea saltern was carried by using time-dependent induction strategy. The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 °C, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid (EDTA), but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.     

Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5?kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4-7) in comparison with Tia (pI 4-5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.     

Hydrolytic action of phospholipase A2 in monolayers in the phase transition region: direct observation of enzyme domain formation using fluorescence microscopy

Phospholipase A2, a ubiquitous lipolytic enzyme highly active in the hydrolysis of organized phospholipid substrates, has been characterized optically in its action against a variety of phospholipid monolayers using fluorescence microscopy. By labeling the enzyme with a fluorescent marker and introducing it into the subphase of a Langmuir film balance, the hydrolysis of lipid monolayers in their liquid-solid phase transition region could be directly observed with the assistance of an epifluorescence microscope. Visual observation of hydrolysis of different phospholipid monolayers in the phase transition region in real-time could differentiate various mechanisms of hydrolytic action against lipid solid phase domains. DPPC solid phase domains were specifically targeted by phospholipase A2 and were observed to be hydrolyzed in a manner consistent with localized packing density differences. DPPE lipid domain hydrolysis showed no such preferential phospholipase A2 response but did demonstrate a preference for solid/lipid interfaces. DMPC solid lipid domains were also hydrolyzed to create large circular areas in the monolayer cleared of solid phase lipid domains. In all cases, after critical extents of monolayer hydrolysis in the phase transition region, highly stabile, organized domains of enzyme of regular sizes and morphologies were consistently seen to form in the monolayers. Enzyme domain formation was entirely dependent upon hydrolytic activity in the monolayer phase transition region and was not witnessed otherwise.     

PURIFICATION AND SOME PROPERTIES OF CELLULASE FROM ODONTOTERMES FORMOSANUS (ISOPTERA: TERMITIDAE)

Abstract  The purification of the cellulase from Odontotermes formosanus workers was achieved by using anion-exchange column of UNOsphere Q, BioLogic DuoFlow chromatography system. The purified cellulase was identified as an endoglucanase and some of its properties were investigated. The EGase activity was 807.5-fold as high as the initial enzyme activity using CMC as substrate and 14.4-fold using salicin as substrate. The enzyme preparations were homogeneous as judged by SDS-PAGE electrophoresis, molecular weight of which was 80 kDa and confirmed by 2-DE zymogram analysis. The enzyme was isoelectric at pH 6.4, which was active on CMC substrate.     

The phospholipase A1 of Trypanosoma brucei does not release myristate from the variant surface glycoprotein

[3H]Myristoyl-labeled variant surface glycoprotein (VSG) has been isolated from Trypanosoma brucei by reverse phase high performance liquid chromatography and used as substrate for the conversion by trypanosomal enzymes of membrane-form VSG to soluble VSG. Conversion is detected by the release of myristoyl-containing lipids. The major lipolytic enzyme of T. brucei, phospholipase A1, is effective for the hydrolysis of myristoyl esters of p-nitrophenol, in a colorimetric assay. However, the phospholipase is unable to cleave the myristoyl ester linkage of VSG. The phospholipase can be separated from the myristoyl-releasing activity of trypanosome homogenate by centrifugation, affinity chromatography, and anion-exchange chromatography. Elution profiles on anion-exchange high performance liquid chromatography also indicate that the phospholipase is inactive against VSG. A small amount of myristoyl-releasing activity associated with the purified phospholipase is probably due to contamination with a phosphodiesterase which releases myristoyl-containing diglyceride from VSG.     

Identification and Purification of Calcium-Independent Phospholipase A2 from Bovine Brain Cytosol

Substantial amounts of phospholipase A2 activity were detected in bovine brain cytosol. The major phospholipase A2 activity was present in the precipitate at 40% saturation with solid ammonium sulfate. After the desaltate of the precipitate was loaded onto an Ultrogel AcA 54 gel filtration column, almost all the activity eluted in the void volume when chromatographed without 1 M KCl. However, when buffer with 1 M KCl was used as the eluent, two active peaks were obtained. One peak (peak I) eluted in the void volume, and the other (peak II) eluted with an apparent molecular mass of 39 kDa as compared with standards. The former was active with diacylglycero-3-phosphoethanolamine, whereas the latter was active with both diacylglycero-3-phosphoethanolamine and 1-alk-1'-enyl-2-acylglycero-3-phosphoethanolamine (plasmenylethanolamine). The apparent molecular mass of peak I was estimated to be 110 kDa as compared with standards on an Ultrogel AcA 34 gel filtration column. Both peaks were purified further with a hydrophobic chromatography column (AffiGel 10 coupled with plasmenylethanolamine) and then by high-resolution liquid chromatography on an MA7Q column. The phospholipase A2 obtained from peak II migrated as one main band with a 40-kDa molecular mass and two minor bands with 14- and 25-kDa molecular masses. Phospholipase A2 obtained from peak I eluted as a single peak on high-resolution liquid chromatography but contained two bands with apparent molecular masses of 100 and 110 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a glucose-6-phosphate isomerase involved in adaptation to salt stress of <Emphasis Type="Italic">Dunaliella salina</Emphasis>

The unicellular green alga Dunaliella salina is a recognized model for studying plant adaptation to high salinity. To isolate some salt-induced proteins at proteomics levels and to identify their expressions at gene levels, algal cells at logarithmic phase cultured in 1.5 and 3.5 M NaCl media were harvested for protein extraction. Solubilized proteins were applied to two-dimensional gel electrophoresis (2-DE) and analyzed by ImageMaster 2D Platinum software. Twenty-one protein spots whose intensities were elevated threefold to 13-fold at 3.5 M NaCl as compared to 1.5 M NaCl were analyzed by matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. One salt-induced protein isolated from the 2-DE gels was identified as a glucose-6-phosphate isomerase (GPI) from D. salina (DsGPI). A full-length cDNA of DsGPI was obtained using rapid amplification of cDNA end technique, and it was shown by heterologous expression to encode a protein with a molecular weight consistent with the protein spot in the 2-DE gels. Real-time quantitative RT-PCR demonstrated that the mRNA of DsGPI was induced up to eightfold (P < 0.01) by 2.5 M and 14-fold higher (P < 0.01) by 3.5 M NaCl than by 1.5 M NaCl, respectively. It is concluded that the protein isolated through 2-DE is indeed DsGPI and that the DsGPI gene may be involved in adaptation to high salinity.     

Amino-terminal and carboxyl-terminal half-molecules of ovotransferrin: preparation by a novel procedure and their interactions

The amino- (N-) and carboxyl- (C-)terminal half-molecules of ovotransferrin were prepared by a novel procedure. The trypsin-nicked ovotransferrin (Ikeda et al. (1985) FEBS Lett. 182, 305-309), in which the two half-molecules interact non-covalently forming a stable dimer, was purified by gel filtration and anion-exchange column chromatography. By subsequent cation-exchange chromatography, the nicked form was distinctly separated into an equivalent amount of the N-terminal and C-terminal half-molecules. Analyses of the N-terminal and C-terminal sequences indicated that the N-terminal and C-terminal half-molecules comprised the alignments of residues 1-332 and 342-686 of ovotransferrin, respectively. Anion-exchange chromatography, gel filtration chromatography, and non-denaturing polyacrylamide gel electrophoresis revealed that the isolated half-molecules had the ability to re-associate in solution. The contents of alpha-helix and beta-sheet of the two half-molecules, as determined by circular dichroism (CD) spectra, were very similar to those of intact ovotransferrin. No prominent alteration in the secondary structure of the two half-molecules was induced by the re-association.     

Improvement of the two-dimensional gel electrophoresis analysis for the proteome study of Halobacterium salinarum

Inherent problems exist in the use of two-dimensional gel electrophoresis (2-DE) for sample preparation and separation of proteins from Halobacterium salinarum. In particular, proteins from cells grown in 25% NaCl are difficult to resolve by 2-DE due to the abundance of salt. To remove salts, a 3 kDa molecular weight cut-off column was used. When soluble proteins were separated by 2-DE, most of the proteins were concentrated in the acidic range. For separation of proteins in the pH 3-6 range, ultrazoom immobilized pH gradient strips were used. In addition, sample separation using a IPGphor/Multiphor combined system was a more effective method for the proteome analysis of acidic proteins than using IPGphor for the isoelectric focusing step.