Polyacrylamide Slab Gel Electrophoresis Research Articles

Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.

We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.

Analysis of esterase zymograms of Fusarium sambucinum and related species.

Esterase zymograms were obtained following polyacrylamide slab gel electrophoresis of protein extracts Fusarium sambucinum and related species originating from different geographic locations and different matrices. The sites of esterase activity were recorded, and the Rfs were calculated. The data were used for the construction of phenograms by cluster analysis and nonlinear mapping by computerized classification techniques. The fifteen isolates of F. sambucinum, the eight isolates of F. torulosum and the six isolates of F. spec. nov. each had identical profiles, and are therefore electrophoretically distinct species. The isolates of F. sarcochroum, one of F. sambucinum sensu lato (BBA 64280) and fifteen isolates of F. sambucinum were electrophoretically indistinguishable from each other. We assume they are synonymous. The isolate of F. bacteridiodes, one of F. sambucinum sensu lato (BBA 64993) and eight isolates of F. torulosum had uniform EST patterns, therefore the two species are electrophoretically identical. We assume they are also synonymous. The remaining three isolates of F. sambucinum sensu lato are somewhat closely related to F. sambucinum isolates on the basis of our investigations.

Role of ascorbic acid in procollagen expression and secretion by human intestinal smooth muscle cells

The role of ascorbate in the production and secretion of procollagen by human intestinal smooth muscle cells and the conditions in culture for optimal ascorbate bioefficacy were studied. Procollagen synthesis and secretion were determined by the incubation of cells with L-[5-3H]proline, and the quantitation of radiolabelled procollagen bands in the cell layer and the culture medium by polyacrylamide slab gel electrophoresis and densitometry. When cells were cultured without ascorbate in the culture medium, procollagen secretion into the medium was 75% less than in cells receiving fresh ascorbate daily. In the cell layer, in contrast, procollagen accumulation was fourfold greater in the scorbutic cells than in the ascorbate-replete cells. These findings contrasted with those in a control line of scorbutic human dermal fibroblasts in which a 95% decrease in procollagen secretion was not associated with any procollagen accumulation in the cells. In the intestinal smooth muscle cells, the absence of ascorbate resulted in a 25 and 50% decrease in steady-state levels of procollagen I and III mRNA, respectively, compared to a 40 and 75% decrease in fibroblasts. Heat inactivation of the serum in the culture medium augmented the promotion of procollagen secretion by ascorbate two- to fourfold. L-ascorbate phosphate did not increase the activity of L-ascorbate when replaced in medium either daily or every 4 days, and its efficacy was not augmented by serum heat inactivation. The changing of culture medium induced collagen secretion in the absence of ascorbate, but this process was markedly enhanced by ascorbate and induced a transient decrease in the steady-state levels of both procollagen and nonprocollagen mRNAs. The predominant action of L-ascorbate on HISM cells in vitro is to promote procollagen secretion and not procollagen synthesis. L-ascorbate-phosphate is not an adequate substitute for L-ascorbate in this cell line.

Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose

A simple method for purification of heat-labile enterotoxin produced by enterotoxigenic Escherichia coli and for purification of cholera toxin using immobilized D-galactose column is described. A single run of column chromatography yielded homogeneous toxin as demonstrated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis.

Electrophoretic Characterization of Multiple Genetic Stocks of Barramundi Perch in Queensland, Australia

We investigated the amount and pattern of genetic variation expressed by the barramundi perch Lates calcarifer throughout Queensland to identify and characterize population subdivision. Fourteen loci known to be variable in this species were screened in 2,912 fish from 24 different areas in Queensland by means of horizontal starch gel and slab polyacrylamide gel electrophoresis. Eleven loci were used in the statistical analysis of stock structure, Seven of these loci (EST-2*, ESTD*, mIDHP*, sIDHP*, LDH-C*, PGDH*, and PROT*) were polymorphic at the 0.95 level (frequency of the most common allele was 0.95 or less) in at least one collection; four loci (IDDH*, FH*, sMDH-A*, and PGM*) were polymorphic at the 0.99 level. Genotype proportions at these 11 loci exhibited close agreement with Hardy–Weinberg expectations. An overall chi-square test of allele frequencies revealed substantial genetic heterogeneity among the 24 collections. Successive rounds of contingency chi-square tests involving geographically adjacent pairs of collections were conducted to identify individual genetic stocks and to locate interstock boundaries. Seven genetically distinct stocks of Queensland barramundi perch were identified by this process. Similar tests demonstrated that Queensland stocks were distinct from neighboring stocks in the Northern Territory of Australia and in Papua New Guinea. The considerable genetic differentiation of barramundi stocks demonstrated by this analysis has important implications for fishery management, aquaculture, and conservation of this species.

Affinity electrophoresis in multisectional polyacrylamide slab gels is a useful and convenient technique for measuring binding constants of aryl sulfonamides to bovine carbonic anhydrase B

This paper describes convenient preparations of heterogeneous multisectional polyacrylamide slab gels and the protocols that use these gels to measure protein-ligand binding constants [using bovine carbonic anhydrase B (CAB) as a model system]. Unlike procedures for affinity electrophoresis using tube gels, all binding information concerning protein-ligand interactions was encoded in a single multisectional gel: the procedure involving for measuring binding constants required no postelectrophoresis manipulation of gels. Use of these types of gels improves the accuracy of affinity gel electrophoresis (AGE) by providing reliable internal protein standards. Binding constants measured by AGE agree with those determined in homogeneous solution by spectrophotometric measurements. This technique has been used to investigate the influence of the length of the spacer separating the ligand and the polyacrylamide backbone on the binding constants. Dissociation constants obtained using the affinity gels approach the values measured in free solution, when the spacer is sufficiently long (> or = 18 A); affinity ligands having short spacers give high apparent dissociation constants.

Characterisation of non-pigmented species of the genus Prevotella by polyacrylamide gel electrophoresis.

Gram-negative anaerobic bacilli previously known as the melaninogenicus-oralis group of Bacteroides have been assigned to a new genus, Prevotella. The non-pigmented members of this genus share several general characteristics and cannot be readily distinguished by routine tests. A polyacrylamide slab gel electrophoresis procedure, with visual analysis of protein patterns, was used to compare cellular protein patterns from clinical isolates with those from collection (reference) strains. Reference strains of P. oralis, P. veroralis, P. buccalis, P. oris, P. buccae, P. zoogleoformans, P. bivia, P. disiens, P. oulora, B. (P.) capillus and B. (P.) pentosaceus, and 91 non-pigmented isolates from patients with adult periodontal disease were examined by conventional biochemical tests, gas-liquid chromatography (GLC) and enzyme tests, and whole-cell protein profiles were obtained by SDS-PAGE. There was close correlation between patterns of results in biochemical and GLC tests and the SDS-PAGE profiles, and the species were readily distinguished in SDS-PAGE. The periodontal isolates were assigned to 10 groups by conventional test reaction patterns and nine groups by SDS-PAGE; the profiles of 79 isolates corresponded to those of seven species reference strains. By SDS-PAGE, clinical isolates of P. buccae (42 isolates) and P. oralis (eight isolates) showed good similarity with reference strains. However, for P. veroralis (15), P. oris (7), P. bivia (4), P. zoogleoformans (2) and P. buccalis (1), clinical isolates showed some minor variations from reference strains. Twelve isolates remained undesignated in SDS-PAGE analysis. Variant SDS-PAGE profiles divided clinical isolates of P. buccae into two subgroups and those of P. veroralis into five subgroups.

Characterization of a 67 kDa microtubule-binding protein in the pancreas from different species

Microtubule-interacting proteins have been studied from a pancreas supernatant. These proteins were first identified by affinity chromatography on taxol-stabilized microtubules. Among these interacting polypeptides, we show, for the first time, the presence of a protein which has a molecular mass of 67 kDa, as determined by polyacrylamide slab gel electrophoresis. The heat stability and the ability of this 67 kDa polypeptide to copolymerize with phosphocellulose-purified tubulin suggest that this protein may be a microtubule-associated protein.

Collagen production by human smooth muscle cells isolated during intestinal organogenesis

The extracellular matrix influences organogenesis by modulating cell behavior. In humans, collagen is the major matrix constituent of the adult intestinal wall and is synthesized by smooth muscle cells. The objective of the current study was to examine collagen production by fetal human intestinal smooth muscle cells isolated during intestinal morphogenesis. Techniques were developed for the isolation and culture of human fetal intestinal smooth muscle cells. The cultured cells were confirmed as muscle by immunohistochemical stains for cytoskeletal filaments and documentation of contractile behavior. In culture, these cells stained for mesenchymal and muscle cytoskeletal proteins: vimentin, actin, and desmin, and did not stain for neural or epithelial markers. The muscle cells contracted in response to acetylcholine, in contrast to human fetal dermal fibroblasts which did not contract appreciably. Collagen production was assayed by the uptake of [3H]-proline into collagenase-digestible protein. Collagen production was greatest at 11 weeks gestation, the youngest age studied. By 20 weeks gestation, collagen production had decreased to adult levels. However, when compared to another matrix-producing fetal mesenchymal cell, the dermal fibroblast, intestinal smooth muscle cells produced twice as much collagen. Collagen types were determined by polyacrylamide slab gel electrophoresis. Smooth muscle cells predominantly produced types I and III collagen alpha chains. Therefore, collagen production is a significant function of human fetal intestinal smooth muscle cells, and probably plays a major role in the development of intestinal structure. The in vitro model presented here provides a means of studying the regulation of this collagen production throughout intestinal organogenesis.

Plasminogen activator: Isolation and purification from Lymphosarcoma of ascites bearing mice

Plasminogen activator secreted by lymphosarcoma (ascites) of mice was purified up to 163-fold by ammonium sulphate fractionation at 35% saturation and chromatography on p-aminobenzamidine-Sepharose 4B. The purified activator contained specific activity of 9980 IU/mg. The plasminogen activator displayed homogeneity by polyacrylamide slab gel electrophoresis and high performance liquid chromatography. The activator consisted of a single polypeptide chain with an apparent molecular weight of 66,000 daltons as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions as well as gel filtration on Sephadex G-100. Distinct differences between this activator and urokinase were discernible in respect of specific activities, fibrin affinity and immunochemical properties. The lymphosarcoma activator appears to be of tissue-type origin since it showed gross similarity to standard tissue plasminogen activator in terms of modes of binding to fibrin and immunological attributes.

Pectic zymograms as criteria in taxonomy of Rhizoctonia

The extracellular pectic enzymes produced by members of the Ceratobasidiaceae were examined by Polyacrylamide slab gel electrophoresis. Cultures included species of Aquathanatephorus, Ceratobasidium, Pseudopapulaspora, Rhizoctonia and Waitea , as well as anastomosis grouped cultures of Ceratobasidium and Thanatephorus . Zymograms were reproducible, little influenced by cultural conditions, and provided, in a permanent record, a means for comparison of isolates. Correlations were found between zymogram patterns and anastomosis groups and their subgroups that have been recognized by morphology and pathology. Zymogram evidence supported some reported inter-relationships, such as between R. cerealis , AG-D and CAG-1. A relationship was found between AG-K and CZG5, also between fusion group E of Richter & Schneider, CAG6, AG-E and R. muneratii . The pectic zymogram method gave promise of aiding practical recognition of species in the complex.

Choline derivatives increase two different acid phosphatases in Rhizobium meliloti and Pseudomonas aeruginosa

In Pseudomonas aeruginosa and Rhizobium meliloti several choline derivatives, utilized as the sole carbon and nitrogen source, increase acid phosphatase activity. The enzyme activity of both bacteria could be released into the surrounding medium by EDTA-lysozyme treatment. The R. meliloti acid phosphatase activity of crude periplasmic extracts measured with p-nitrophenylphosphate was not inhibited by the presence of 5 mM choline, betaine, trimethylammonium or phosphorylcholine. The activity could not be detected using phosphorylethanolamine or phosphorylcholine as substrates. Among several phosphoesters tested only pyridoxal-5-phosphate was hydrolyzed at a considerable rate. In 7.5% polyacrylamide slab gel electrophoresis (non-denaturing conditions) of crude extracts obtained from bacteria grown in the presence of serine, glutamate, aspartate or dimethylglycine a phosphatase activity with identical mobility could be detected with alpha-naphthylphosphate or pyridoxal-5-phosphate were used as substrates. In conclusion, although the choline metabolites are capable of increasing acid phosphatase activities in R. meliloti and in P. aeruginosa, there are two different enzymes involved, apparently in different metabolisms.

Enzymes of strains of pleurotus species (Basidiomycetes) compared by electrophoresis.

Four kinds of electrophoresis were used to characterize enzymes in 11 isolates from six species in the genus Pleurotus (Fr.) Quel: Pleurotus ostreatus, P. pulmonarius, P. spodoleucus, P. salmoneostramineus, P. cornucopiae, and P. cystidiosus. They were agarose isoelectric focusing (IEF), polyacrylamide IEF, 7.5% polyacrylamide gel electrophoresis (PAGE) and 4-15% gradient PAGE. Ultimately, the 4-15% polyacrylamide gel-slab electrophoresis was found to be most suitable for our study in numerically analyzing the patterns of seven enzymes. The enzymes were 6-phosphogluconate dehydrogenase (EC 1.1.1.41 6PGDH), malate dehydrogenase (EC 1.1.1.37 MDH), lactate dehydrogenase (EC 1.1.1.27 LDH), glutamate dehydrogenase (EC 1.4.1.4 GDH), glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH), alcohol dehydrogenase (EC 1.1.1.1 ADH), and esterase (EC 3.1.1.1 Est). The isolates were compared by using an index of similarity (Sm) based on relative mobilities (Rm) of the enzyme bands. By this method, the isolates were reasonably grouped as species. Based on the Sm values, as well as intercompatibilities and morphological characters, the relationships between P. ostreatus, P. spodoleucus and P. pulmonarius were analyzed.

Identification of Different Forms of Human C4b-Binding Protein Lacking β-Chain and Protein S Binding Ability

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

Characterization of thermostable invertase from wine grapes

The levels of invertase activity were found to vary significantly among 14 varieties of grapes tested. The crude invertases were, however, similar in both pH and temperature optima, as well as pH and thermal stabilities. The enzymes showed a high degree of thermostability and were also stable at acidic pH and high concentration of alcohol. A significant level of invertase activity persisted in several white table wines. The optimum activity of the enzyme purified from Semillon grape was observed at about 75°C and it was stable up to 70°C. Although the enzyme was stable between the pH 2 and 8, the optimum pH for its activity was about 4. The enzyme, whose molecular weight was estimated to be 65,000 by SDS polyacrylamide slab gel electrophoresis, was found to be a glycoprotein with a total carbohydrate content of 33%. This enzyme showed activity toward sucrose and raffinose, but was inactive on the other disaccharides tested.

Isolation and characterization of hemorrhagic, myonecrotic and edema-inducing toxins from Bothrops insularis (jararaca ilhoa) snake venom

Bothrops insularis snake venom was fractionated by gel filtration on Sephadex G-150 followed by ion-exchange chromatography on SP-Sephadex C-25. Two active fractions were purified to homogeneity: (1) SIII-SpI, approximate mol. wt 32,000 and N-terminal amino acid residue Val. This fraction showed esterase activity on TAME, edema-inducing activity on the rat hind paw and contractile activity on the isolated guinea pig ileum. The latter two activities were antagonized by benadryl plus methysergide; (2) SIII-SpVI, a myonecrotic and edema-inducing phospholipase, approximate mol. wt 29,000, N-terminal amino acid residue pyro-Glu, consisting of two chains of approximately 15,000 mol. wt each linked by disulphide bridge(s). The induction of edema by this fraction was not antagonized by benadryl plus methysergide, indomethacin, BW755C or BN52021, but it was antagonized by dexamethasone. Three highly purified hemorrhagic heterodimeric fractions, SIII-SpIII-3, SIII-SpIII-4 and SIII-SpIII-5, of approximate mol. wts 26,000, 29,000 and 26,000, and having N-terminal residues of Asx, Asx and Gly, respectively, were further isolated by preparative polyacrylamide slab gel electrophoresis. SIII-SpIII-4 and SIII-SpIII-5 increased the recalcification time of citrated rat plasma. None of the five isolated fractions showed any proteolytic (on casein) or kininogenase activity.

Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates ofVibrio hollisae

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.

Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates of Vibrio hollisae

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.

Purification and Properties of UDP-Glucuronyltransferase from Kidney Microsomes of β-Naphthoflavone-Treated Rat

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)

Glyceraldehyde-3-phosphate dehydrogenase is a microtubule binding protein in a human colon tumor cell line.

A protein which binds to tubulin polymer was isolated from a human colonic tumor cell line. This protein has a molecular mass of 35 kDa, as determined by polyacrylamide slab gel electrophoresis. The protein was purified by affinity chromatography on taxol-stabilized microtubules, and it did not cross-react with anti-MAP2 or anti-tau antibodies. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase by its enzyme activity and immunoblotting experiments. The purified protein caused a pronounced enhancement in the turbidity increase produced by in vitro tubulin polymerization, and electron microscopic observations revealed the presence of bundles of microtubules.