Genetic identification of rat liver carboxylesterases isolated in different laboratories

Six carboxylesterases previously isolated from rat liver microsomes, characterized in Brussels and in Kiel, were compared with genetically defined liver esterases of various reference strains using polyacrylamide gel electrophoresis and isoelectric focusing. The six liver carboxylesterases were identified as alloenzymic forms of ES-3, ES-4, ES-8/ES-10 and ES-15 according to the genetic nomenclature recommended by van Zutphen (Van Zutphen, L.F.M. (1983) Transplant. Proceed. 15, 1687-1688). The genetic and biochemical characteristics of the four isoenzymes are summarized, and their identity with several other drug-metabolizing esterases/amidases and lipases of rat liver endoplasmic reticulum is discussed.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

Multiple molecular forms of soluble esterases in the digestive system of the developing chicken

Soluble esterases in tissues of endodermic origin and of various developmental ages of Gallus gallus were analysed by polyacrylamide slab gel electrophoresis and characterized as carboxylesterases, acetylesterases and cholinesterases. Esterase bands were observed from day 9 in the liver and from day 6 in stomach, intestine and yolk sac. The electrophoretic profiles became more complex after hatching with concomitant increase in the staining intensity. On isoelectric focusing of liver extracts only a major form with pI 5.4 was observed. An eserine sensitive band designated EL-1 was found to be tissue (liver) and age (upon hatching) specific. EL-1-like isozymes were also observed in other species of the Galliformes order.     

Effects of low temperature on wheat leaf proteins

Finnish wheat cultivars, winter wheat ‘Vakka’ and spring wheat ‘Apu’, grown for 9 days at 25° and then for 52 days at 2–4°, were called ‘hardened’. Proteins and esterases of young leaves were resolved by polyacrylamide gel electrophoresis (PAGE), porosity gradient PAGE (poroPAGE), isoelectric focusing (PAGIF) between pH 3 and 9, and by Na-dodecylsulfate (SDS)-PAGE. Hardened and unhardened leaves have different protein patterns after PAGE, PAGIF, poroPAGE and SDS-PAGE, respectively, as well as for esterases after PAGE and PAGIF. The PAGIF patterns of esterases show distinct changes of two bands especially for hardened leaves of winter wheat, one appearing, the other one disappearing.     

Structural Proteins of Simian Virus 40

Sodium dodecyl sulfate acrylamide gel electrophoresis of the solubilized proteins from purified simian virus 40 (SV40) virions revealed two major and two minor structural polypeptide components. The major components which comprise over 75% of the total virion were shown to be the capsid proteins by immunological and isoelectric focusing fractionation analysis. These two polypeptides have estimated molecular weights of 45,000 daltons as determined by gel electrophoresis. One of the two minor components was identified as the nucleocapsid protein and has an approximate molecular weight of 16,000. The other unidentified minor component has an average molecular weight of 29,000.     

Changes in two ribonuclease isozymes during rust infection of flax cotyledons

A ribonuclease fraction previously purified from flax by gel filtration was further resolved into two components by hydroxyl apatite chromatography. These were homogeneous with respect to electrophoresis and isoelectric focusing. Both enzymes are of RNase I type but differ in substrate specificity, kinetic properties, pH response, and isoelectric point.     

Biochemical and physiological studies of soluble esterases fromDrosophila melanogaster

Twenty-two soluble esterases have been identified inD. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Cholineand acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.This study was funded in part by the Rural Credits Development Fund.     

Two-dimensional polyacrylamide gel electrophoresis of the protease SP220K, a renal cell carcinoma marker

The present study analyses, by two-dimensional polyacrylamide gel electrophoresis, the protease SP220K isolated from renal cell carcinoma. The pure molecule is separated using either immobilized pH gradient or isoelectric focusing in conventional carrier ampholyte in the first dimension. Some interactions with the acrylamide matrix in isoelectric focusing are discussed. The results demonstrate that two-dimensional gel electrophoresis performed with enriched media such as basolateral membranes, allows the detection of the protease. In addition, the non detection of the molecule up to now by this methodology can be explained by the high tendency of oligomerization of SP220K. Effectively the high molecular weight form of the molecule of 220 kDa is favoured in two-dimensional gel electrophoresis over monomeric forms which are better detected in SDS PAGE. This was confirmed by immunostaining performed with an antiserum to SP220K produced by nitrocellulose-bound antigen.     

Purification of soluble murine transplantation antigens by isoelectric focusing

Hydrosoluble transplantation antigens were prepared from membranes and microsomes of C(3)H mice (BP8 tumoral cells) and purified by isoelectric focusing. A biologically active fraction which seems homogenous by acrylamide gel electrophoresis was characterized: it specifically inhibits anti C(3)H hemagglutinin antibodies and provokes a highly significant prolongation of skin graft.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

Electrophoresis and electrofocusing of rhodopsin solubilized by triton X-100]

Detergent-rhodopsin micells (component I) were separated from other fast and slow migrating protein components under electrophoresis of triton X-100 solubilized bovine rod outer segments (ROS). Treatment of ROS by alum caused a complete disappearance of non-rhodopsin proteins and the appearance of slow migrating band (component II). Preliminary bleaching of dark extracts did not affect the migration rate of the component I. The addition of urea to solubilizing mixture caused the increase of component I content and the diffusity components I and II bands. The rate of electrophoretic migration and the content of components I and II sharply decreased together with the appearance of fast migrating pink-brown band after the addition of 2-mercaptoethanol. The extracts from alum-treated ROS were separated into 15-20 protein bands under acrylamide gel isoelectric focusing. Such protein heterogeneity probably depended on the ability of triton X-100 to form micells with different isoelectric points during the interaction with ampholines in the electric field. These micells, having different isoelectric points, are shown to contain one and the same protein--opsin.     

Esterases in quail (Coturnix coturnix). Physicochemical and developmental aspects

• 1.1. Soluble esterases of digestive system organs of various developmental stages in the quail (Coturnix coturnix) were resolved by polyacrylamide gel electrophoresis into several molecular forms which were characterized as carboxylesterases, acetylesterases, cholinesterases and esterases sensitive to eserine.
• 2.2. The pI of the majority of esterasic activity in several quail and chicken tissues was observed in the range of 5.1–5.6, while the apparent molecular weight in liver extracts was 60,000.
• 3.3. The expression of the esterase multiple molecular forms was found to be both tissue- and developmental stage-specific, with electrophoretic patterns becoming more complex in number and/or staining intensity upon hatching and thereafter, especially in liver and intestine.

     

Isoelectric focusing and two-dimensional electrophoresis of tubulin using immobilized pH gradients under denaturing conditions

Modifications of the LKB Immobiline isoelectric focusing (IEF) technique are described for use under conditions that solubilize and denature most proteins (8 M urea and 2% Nonidet-P40). This procedure permits pH gradients that are four- to fivefold shallower than previously available with conventional ampholine-IEF procedures. It can also be used as a first dimension in two-dimensional gel electrophoresis. The advantage of the stable ultranarrow pH gradient is demonstrated by directly comparing the resolution of vertebrate brain tubulins using (i) denaturing conventional ampholine-IEF and (ii) denaturing Immobiline-IEF. Analysis of tubulin on the Immobiline-IEF gel increases the separation distance between the individual tubulins and distinguishes differences among tubulin samples that could not be resolved by conventional ampholine isoelectric focusing.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Polyacrylamide gel electrophoresis: reaction of acrylamide at alkaline pH with buffer components and proteins

The chemical reaction of monomeric acrylamide with primary, secondary, and tertiary amines, used as buffer components in polyacrylamide gel electrophoresis systems, was investigated in the basic pH range. Adduct formation proceeded for several minutes up to weeks, depending on the reactivity of the amino groups. A pH shift in the reaction mixture due to an altered pK value of the reaction product was observed. However, a few primary amines (tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1,3-propanediol) and secondary amines 3-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid, 3-(dimethyl(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid) showed negligible shifts of pH. They are, therefore, useful as components in the polymerization mixture; whereas some tertiary amines showing complete pH stability as well (e.g., triethanolamine) are not suitable, as they acted as accelerators of gel polymerization. Acrylamide can also covalently bind to proteins by reacting with the epsilon-amino group of lysine residues, especially. Bovine serum albumin, having an acidic isoelectric point, and the basic protein cytochrome c were treated with different acrylamide concentrations at alkaline pH yielding modified protein molecules with altered electrophoretic mobilities in different polyacrylamide gel electrophoresis systems. This reaction gave rise to artifacts in alkaline polyacrylamide gels and isoelectric focusing systems when residual acrylamide monomers were still present in the gel matrix after the polymerization process ceased.     

A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution

A highly porous and efficient discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis system is described. The slab get consists of two porous layers of acrylamide of the following composition: 4% acrylamide, 0.04% bisacrylamide for the stacking gel, and 10% acrylamide, 0.1% bisacrylamide for the separating gel, both layers having different buffers. The separating gel mixture (final pH 9.0) and the buffers of the electrode chamber (pH 8.45) consist of Tris and glycine in such a ratio that no acid or base is necessary to adjust the pH. The resulting gel system has the following advantages: (a) it is able to resolve the components from large-volume samples (up to 200 microliter) after an overnight electrophoresis run while still maintaining the capacity to produce very sharp bands; (b) it has a high and broad resolution, allowing the separation on the same gel of proteins with apparent molecular masses between 10,000 and 450,000 Da; (c) it is very easy to prepare and shows excellent reproducibility in the electrophoretic patterns; (d) when used as a second dimension in tandem with isoelectric focusing, it improves the resolving power of two-dimensional gel electrophoresis; and finally, (e) its low crosslinker-to-acrylamide ratio allows the effective and rapid transfer of proteins to nitrocellulose membrane, thus improving the usefulness of protein blotting. In all cases, adrenal medullary chromaffin cell proteins were used as test samples.     

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Phosphodiesterase from the venom of Crotalus ruber ruber

Phosphodiesterase was isolated from the venom of Crotalus ruber ruber from the U.S.A. using the gel filtration on a Sephadex G-75 column, followed by anion or cation exchange chromatography. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and isoelectric focusing electrophoresis. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB), but not by soybean trypsin inhibitor (SBTI) or benzamidine. The molecular weight of this enzyme was determined to be approx. 98,000 and the isoelectric point was found to be pH 10.5 by isoelectric focusing with carrier ampholyte. This enzyme contained 1.04 mol zinc per mol. The Michaelis constant (Km) of this enzyme for p-nitrophenyl thymidine-5'-phosphate and inhibition constant (Ki) for PCMB were found to be 8.3 X 10(-3) and 1.2 X 10(-2) M, respectively.