Ultrathin-layer Isoelectric Focusing Research Articles

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

Ultrathin-layer isoelectric focusing for bovine serum transferrin phenotyping

High resolution of human lactate dehydrogenase: new multiple forms and potential tumor markers.

Human lactate dehydrogenase (LDH) was investigated by ultrathin-layer polyacrylamide gel isoelectric focusing (IEF). In serum, approximately 15 bands and in lyzates of erythrocytes approximately 20 bands were detected. Known LDH isoenzymes (identified by markers) appeared in the zymograms as follows: LDH-1 as a single or double band, LDH-2 as a single band in serum and in the marker, and as a double band in hemolyzate, LDH-3 as a double band, and LDH-4 and LDH-5 each as a single band. LDH-1 was partly inactivated, probably due to deamidation in the acidic range of the pH gradient. Potential LDH tumor markers were detected in different tumor cytosols.

Application of Neuhoff's optimized Coomassie Brilliant Blue G‐250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.

The esterase D polymorphism by ultrathin layer isoelectric-focusing: Frequency of the EsD5 allele in the population of Rome

The distribution of EsD phenotypes in the population of Rome was investigated by cellulose-acetate electrophoresis and isoelectric-focusing. The gene frequencies were found to be: EsD1 = 0.8451, EsD2 = 0.1363, EsD5 = 0.0186. These frequencies were compared with those reported in other populations.

Genetic study of red cell esterase D polymorphism by ultrathin layer isoelectric focusing. Distribution in the Veneto population (Italy).

Human red cell Esterase D (EsD) was analyzed by isoelectric focusing (IEF) on ultrathin-layer polyacrylamide gel with a pH range of 5.0-6.0. Hemolysates were treated with Dithiothreitol to avoid loss of activity and change of the isozyme patterns by in vitro storage effects. In our sample of 951 unrelated persons from Veneto, seven different phenotypes were observed. The following allele frequencies were calculated: EsD1 = 0.8476, EsD2 = 0.1336, EsD5 = 0.0178, and EsDV = 0.0010.

Properties of β-glucosidase from Carica papaya fruit

The properties of β-glucosidase from Carica papaya fruit pulp, purified to homogeneity on ultrathin-layer isoelectric focusing (pI, 5·2), were studied. The molecular mass was determined to be 54 000 by gel filtration, and 27 000 by sodium dodecylsulphate polyacrylamide gel electrophoresis, respectively, indicating that the enzyme was composed of two subunits. The optimum pH and temperature for enzyme activity were at 5·0 and 50°C, respectively. The enzyme catalyzed the hydrolysis of aryl-β- d-glucosides and, to a lesser extent, alkyl-β- d-glucosides; disaccharides were hydrolyzed very slightly. Glucosyltransferase and glycosidic ‘side-activities’ (β-galactosidase, β-xylosidase, β-fucosidase and α-arabinosidase activities) were absent. The enzyme was activated by Ca 2+, Mn 2+, Mg 2+ and EDTA and was strongly inhibited by Ag + and Hg 2+. d-Glucono-1,5-lactone (K i, 0.08 m m), 1-deoxy- d-glucose (K i, 6 m m), 1-deoxy-1-amino-β- d-glucose and glucal (K i, each 8 m m) as well as N-methylglucamine (K i, 17 m m) exhibited reversible inhibitory effects. The amino acid composition, as well as sugar content and composition of the enzyme, were also determined. The carbohydrate content of 10% consisted mainly of arabinose (48%) and fucose (23%).

Group-specific Component Subtyping by Isoelectric Focusing in Narrow Range Ultrathin-layer Polyacrylamide Gels Containing a Mixture of Separators

Ultrathin-layer isoelectric focusing in polyacrylamide gels containing a combination of the separators 3-( N-Morpholino)propanesulphonic acid (MOPS) and N-2-Hydroxyethylpiperazine N′-2-ethanesulphonic acid (HEPES) with narrow range Pharmalytes (pH 4·5–5·4) has been used to identify the six common group-specific component (Gc) phenotypes in bloodstains. An improvement in the separation between the Gc1F and Gc1S bands has been achieved over that previously reported for ultrathin-layer gels and which compares favourably with the separation of GclF and Gc1S bands in Immobiline gels.

The polymorphism of plasminogen (PLG) by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy).

The distribution of plasminogen phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 1325), the three common phenotypes PLG1, PLG2, PLG2-1 and two further phenotypes PLG1-V and PLG2-V were, observed and the following frequencies calculated: PLG1 = 0.84038; PLG2 = 0.15811; PLGV = 0.00151. These gene frequencies are compared to those found in other populations. Analysis of 41 mother-child pairs was in agreement with an autosomal codominant inheritance.

Isolation, Purification, and Characterization of Lipase Isoenzymes from a Technical Aspergillus niger Enzyme

ABSTRACTA qualitative screening revealed the occurrence of lipase, esterase, protease, amylase, endo‐1,4‐β‐D‐glucanase, xylanase, pectinmethylesterase, polygalacturonase, catalase, β‐D‐glucosidase and β‐D‐galactosidase activities in the technical Aspergillus niger enzyme under study (Lipase 2212 D, Röhm). The isolation and purification of lipolytic activities were performed by combination of DEAE‐Trisacryl M ion exchange chromatography, Sephadex G 50 gel filtration and hydrophobic chromatography using Phenylsepharose CL‐4B. The individual purification steps were checked by specific enzyme visualization in ultrathin agar gels after ultrathin‐layer isoelectric focusing (UIEF). Two UIEF homogeneous lipase isoenzymes (I and II) were isolated and characterized by the following parameters: isoelectric points (I: 4.0; II. 3.5); molecular weights (I: 31000 daltons; II: 19000 daltons); carbohydrate contents (I: 6%; II: 9%) and compositions; pH optima (I, II: 5‐6); substrate specificities and various effectors.

Biochemical genetics in the yellow wagtail complex: Conservative avian protein evolution reconfirmed by isoelectric focusing

Seventeen protein loci were examined by ultrathin layer isoelectric focusing in three subspecies of the problematic Motacilla flava complex. The percentage of polymorphic loci is 41. Mean heterozygosity per locus and individual ranges from 0.063 to 0.150. Nei's genetic distance ranges from 0.0034 to 0.0054, which is within the range previously reported for other avian subspecies-relations. The genetic differentiation pattern coincides well with morphological and ethological differentiation within this complex and even with the amount of interbreeding of its members in contact zones. Peculiarities of avian evolution are discussed.

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 μU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.

Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase

The phytopathogenic enterobacterium Erwinia chrysanthemi excretes multiple isozymes of the plant tissue-disintegrating enzyme, pectate lyase (PL). Genes encoding PL were cloned from E. chrysanthemi CUCPB 1237 into Escherichia coli HB101 by inserting Sau3A-generated DNA fragments into the BamHI site of pBR322 and then screening recombinant transformants for the ability to sink into pectate semisolid agar. Restriction mapping of the cloned DNA in eight pectolytic transformants revealed overlapping portions of a 9.8-kilobase region of the E. chrysanthemi genome. Deletion derivatives of these plasmids were used to localize the pectolytic genotype to a 2.5-kilobase region of the cloned DNA. PL gene expression in E. coli was independent of vector promoters, repressed by glucose, and not induced by galacturonan. PL accumulated largely in the periplasmic space of E. coli. An activity stain used in conjunction with ultrathin-layer isoelectric focusing resolved the PL in E. chrysanthemi culture supernatants and shock fluids of E. coli clones into multiple forms. One isozyme with an apparent pI of 7.8 was produced at a far higher level in E. coli and was common to all of the pectolytic clones. Activity staining of renatured PL in sodium dodecyl sulfate-polyacrylamide gels revealed that this isozyme comigrated with the corresponding isozyme produced by E. chrysanthemi. The PL isozyme profiles produced by different clones and deletion derivative subclones suggest that the cloned region contains at least two PL isozyme structural genes. Pectolytic E. coli clones possessed a limited ability to macerate potato tuber tissues.

Trennung multipler Enzymformen durch isoelektrische Fokussierung in ultradünnen Polyacrylamidgelschichten auf Polyesterfolie

Summary Multiple forms of barley leaf enzymes (peroxydase, superoxid dis mutase, esterase, acid phosphatase, and leucin aminopeptidase) were determined using ultrathinlayer isoelectric focusing in 0,18 mm thick polyacrylamide gels on polyester films. The ultrathinlayer isoelectric focusing turns out to be an excellent method for the separation of isoenzymes. The precision of the methods used allows statistical plots of the results. Staining procedures were adapted to other conditions in ultrathin gels.

Glycosylation of human albumin in diabetes mellitus II. Extensive in vitro modification by trioses and hexoses as revealed by isoelectric focusing

AbstractPurified human serum albumin (HSA) from normal adults has been reacted in vitro with different trioses and hexoses and the reaction products analysed by ultrathinlayer isoelectric focusing (IEF) under native and denaturing (8 M urea) conditions. The order of reactivity of these molecules has been found to be: glyceraldehyde > glyceraldehyde 3‐phosphate > glucosamine ≫ glucose ≫ acetaldehyde. By chemical analysis, the number of residues of glyceraldehyde bound to albumin appears high: 130 moles/mole of protein, while the number of exposed lysines is halved: ca. 8 in reacted vs. 16 in control albumin. It appears that a minimum length for an aldehyde to react with protein amino groups is three carbon atoms. A completely different IEF behavior has been found between “reacted and NaCNBH4 reduced” vs. “reacted and rearranged” HSA molecules. The former group, even when extensively reacted with sugars, exhibits a normal surface charge at pH = pI, possibly because, at the acidic pIs of HSAs, the now secondary (sugar‐bound) amino groups are still fully protonated. In the latter group, the bound sugar spontaneously undergoes an Amadori rearrangement followed by secondary reactions, still not fully clarified, which could possibly lead to ring structures (e. g. pyrrolic) with complete loss of positive charge from the sugar‐reacted amino groups. These species, in fact, show a markedly more acidic pI.

Multiple crossed immunoelectrophoresis on agarosecoated GelBond film

AbstractA new technique, multiple crossed immunoelectrophoresis on agarose‐coated GelBond film, is described. A sheet of GelBond with agarose areas containing a single or different antisera can be used for the simultaneous analysis of eight samples. Antigens were separated in the first dimension (i) in an inert medium according to net charge (cellulose acetate membrane elecrophoresis), (ii) in a sieving medium according to charge and molecular size (ultrathin‐layer polyacrylamide gradient gel electrophoresis), or (iii) in a pH gradient according to differences in isoelectric points (ultrathinlayer isoelectric focusing). By using these methods in the first dimension, resolution is improved over that in conventional agarose electrophoresis. The first dimensional strips are easily transferred to the second dimensional antibody‐containing agarose. Use of the GelBond film allows easy handling and convenient storage. Multiple crossed immunoelectrophoresis is a versatile method for rapid analysis of antigenic variability.

Proteins and Peroxidase in Callus and Suspension Cultures of Apple

Different methods for the isolation of soluble proteins were applied to cell cultures of three apple cultivars (Malus sylvestris Mill.), best results being obtained with a rapid technique based on freezing and thawing. Ultrathin-layer isoelectric focusing followed by an improved silver staining method has shown that proteins from apple callus cultures consist of some 60 to 80 zones, with isoelectric points mainly between pH 4 to 7. Depending on protein content, adequate silver staining is achieved with 50 to 500 cells. Protein patterns of callus cultures allowed clear discrimination of cultivars. Protein and peroxidase isozyme patterns in cell saps of suspension cultures show striking differences during the growth cycle, whereas the protein patterns from the nutrient media were constant over the entire cultivation period and closely resembled the patterns of stationary phase and callus cells.

Silver staining of immunofixed group specific component on cellulose acetate membranes after isoelectric focusing in narrow pH interval gels

AbstractThe sensitivity of group specific component (Gc) detection by immunofixation after isoelectric focusing (IEF) has been examined by using two IEF methods: ultrathinlayer IEF and immobilized pH gradient (IPG) focusing followed by two staining methods: Ponceau S and a combination of Ponceau S with silver staining (SS). Both diluted plasma and bloodstain extracts were examined by these methods at two dilutions of immunofixation antibody ‐ 1:6 and 1:24. The cellulose acetate membranes containing the Gc precipitates were washed with sodium chloride after the SS process. This greatly improved the visibility of the Gc bands. The method was found to be more sensitive than Ponceau S and its use reduced the cost of immunofixation by 75%. Ultrathin‐layer IEF was carried out in gels containing the pH 4.5–5.4 Pharmalytes. The separator N‐2‐hydroxyethylpiperazine‐N′ 2‐ethanesulfonic acid (HEPES) was added to some gels and the effect on the resolution of the Gc 1 subtypes was examined and compared with the separation achieved with IPG gels. It was found that ultrathin‐layer IEF using narrow range Pharmalytes and the separator HEPES was the more sensitive method; however, IPG gels were easier to interpret.

Gc subtypes determined by ultrathin-layer isoelectric focusing. Distribution in the Veneto population (Italy).

The distribution of Gc phenotypes in the population of Veneto was investigated by ultrathin-layer isoelectric focusing. In our sample (n = 732) the six common phenotypes, Gc 1S, 1F, 1S1F, 2, 2-1S, 2-1F and a further phenotype, GC 1S1C3, were observed and the following frequencies calculated: Gc 1S = 0.560792; GC 1F = 0.159153; Gc2 = 0.277323; Gc 1C3 = 0.002732. Our gene frequencies have been compared with those found in other populations.

The PGM 1 polymorphism as revealed by ultrathin-layer isoelectric focusing in the population of Padua

The occurrence of PGM 1 phenotypes in 589 samples from the population of Padua was investigated by ultrathin-layer isoelectric focusing. All ten phenotypes were observed. Frequencies of the PGM 1 alleles (1+ = 0.6180; 1− = 0.1163; 2+ = 0.2122; 2− = 0.0535) have been compared to those found in other populations.

The polymorphism of transferrin by ultrathin-layer isoelectric focusing. Tf phenotypes and TfC subtypes in the population of Padua

The distribution of Tf phenotypes in the population of Padua was investigated by ultrathin-layer isoelectric focusing. In our sample ( n = 618) nine phenotypes, Tf C 1, C 2, C 3, C 3−1, C 2−1, C 3−2, C 1B, C 2B and C 1D, were observed and the following frequencies calculated: TfC 1 = 0.77837; TfC 2 = 0.1804; TfC 3 = 0.03641; TfB = 0.0040; TfD = 0.0008. These gene frequencies have been compared to those found in other populations. Analysis of 101 mother-child pairs was in agreement with an autosomal codominant mode of inheritance.