Focusing In Thin-layer Polyacrylamide Gels Research Articles

Characterization of adult Dicrocoelium dendriticum by isoelectric focusing.

Water soluble extracts of 3131 adult specimens of Dicrocoelium dendriticum from cattle, sheep and goats, mainly from León province, were analysed by isoelectric focusing in thin-layer polyacrylamide gels. Activity of the following enzymes was studied: lactate dehydrogenase (LDH, EC 1.1.1.27), glucose phosphate isomerase (GPI, EC 5.3.1.9), phosphoglucomutase (PGM, EC 2.7.5.1), acid phosphatase (AcP, EC 3.1.3.2), alpha-glycerophosphate dehydrogenase (alpha-GPDH, EC 1.1.1.8), hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.30) and malate dehydrogenase (MDH, 1.1.1.37). Five distinct enzyme types were recognized for LDH (pH range 6.30-7.13), GPI (pH 6.13-6.80) and PGM (pH 6.20-6.60) whereas AcP showed three different patterns (pH 5.70-5.92). Weak and diffuse activity was detected for MDH (pH 4.8-6.2) and no activity was observed for alpha-GPDH and HBDH. In general, little phenotypic variation was observed between worms recovered from a single host, between those from hosts of the same species and between those from hosts of different species, although some enzyme types were found in some animals but not others. Nevertheless, it must be taken into account that most parasites came from sheep and also from a relatively small area in north-west Spain.

Using Isozyme Polymorphisms for Identifying and Assessing Genetic Variation in Cultivated Pawpaw [Asimina triloba (L.) Dunal

The utility of isozyme phenotypes for identifying and determining genetic variation in pawpaw cultivars was studied using isoelectric focusing in thin-layer polyacrylamide gels. Based on a sample of 32 clones (cultivars and advanced selections) and 23 enzyme systems, 7 enzymes were found to be polymorphic, involving 9 polymorphic loci [acid phosphatase (ACP), dihydrolipoamide dehydrogenase (DDH), malic enzyme (ME), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), peroxidase (PRX), and shikimate dehydrogenase (SKD)]. Altogether these 9 loci and 32 clones yielded 28 multi-locus isozymic phenotypes useful for cultivar identification; 24 of the 32 clones were uniquely identified. The allozyme variation in these clones has the average of other long-lived woody perennials of widespread geographic range in temperate regions with insect-pollinated outcrossing breeding systems, secondary asexual reproduction, and animal-dispersed seed. Genetic differentiation among these pawpaw clones, measured by Nei's distance, D, was substantial: 496 pairwise comparisons of genetic distance among the 32 clones indicated that they differed on average of D = 0.068 ± 0.04 and ranged from 0 to 0.188. Cluster analysis (UPGMA) produced a most likely division of the 32 clones into 7 groups; however, these groups did not conform to known pedigree relations. Additional polymorphic enzymes are needed for accurate allozyme-based genetic discrimination.

Low frequency of PIM3 gene in patients with monoclonal gammopathies.

The distribution of PI (protease inhibitor) phenotypes and PI M subtypes was studied in 200 patients with monoclonal gammopathies and 320 healthy blood donors by isoelectric focusing in thin-layer polyacrylamide gels, pH range 4-5. The distribution of PI phenotypes and gene frequencies in the patients differed significantly from the corresponding values found in the blood donors, as shown by the chi 2 test (chi 2 = 16.5, p = 0.02 and chi 2 = 17.6, p = 0.0014, respectively). A lower frequency of the M3 variant was observed in patients, both in homozygous (PI M3) and heterozygous forms (PI M1M3 and PI M2M3). No significant difference between PIz allele frequencies in patients and healthy controls was found.

Application of Isoelectric Focusing in Thin-Layer Polyacrylamide Gels to the Study of Opine Dehydrogenases in Marine Invertebrates

Application of Isoelectric Focusing in Thin-Layer Polyacrylamide Gels to the Study of Opine Dehydrogenases in Marine Invertebrates

Genetic studies on human thyroxine-binding globulin (TBG)

An enzyme immunoassay technique combined with Western blotting is described to demonstrate thyroxine-binding globulin (TBG) by isoelectric focusing in thin-layer polyacrylamide gels with 8 mol/l urea. Quantitative evaluation was by laser densitometry. No genetic charge variants of TBG were encountered in a sample of 840 unrelated individuals from southwestern Germany. There was no correlation between structural and quantitative variations in the TBG protein. Results from a family with quantitative TBG deficiency strongly support the postulated X-linked mode of inheritance. The method described can be considered as an additional diagnostic tool in thyroid evaluation.

Plasma protein and red cell enzyme groups in Galicia (north west Spain).

Galactose-1-phosphate uridyl transferase (GALT), esterase D (EsD), and plasminogen (PLG) phenotypes were determined by isoelectric focusing in thin-layer polyacrylamide gels (PAGIF) in a random sample from Galicia. Haptoglobins (Hp) were determined by conventional electrophoresis. The following gene frequencies were observed: for GALT: GALTN: 0.930; GALTD1: 0.044; GALTD2: 0.025; for EsD: EsD1: 0.874; EsD2: 0.104; EsD3: 0.021; for PLG: PLG1: 0.800; PLG2: 0.199; for Hp: Hp1: 0.426; Hp2: 0.573. Population data results of all electrophoretic markers typed until now in Galician population are also included.

Distribution of α<sub>2</sub>HS-Glycoprotein Types in Egyptian

The polymorphism of α2HS-glycoprotein (α2HS system) was stu diedin a total of 95 unrelated Egyptian individuals living in Tanta city, Gharbiya province, Nile Delta of Egypt, using isoelectric focusing in thin-layer polyacrylamide gel and immunofixation. Three common phenotypes, α2HS1, 2-1 and 2 were demonstrated and the estimated allele frequencies were α2HS1=0.86 and α2HS2=0.14.

Red cell phosphoglucomutase (PGM 1) subtypes in Egyptians

The polymorphism of the human red cell phosphoglucomutase 1 (PGM 1) in samples from Egyptians ( n = 134) was investigated using isoelectric focusing in thin-layer polyacrylamide gel. In the studied population samples nine common phenotypes were observed, and the calculated frequencies for the genes PGM 1 1+, PGM 1 1−, PGM 1 2+ and PGM 1 2− were 0.6381, 0.0821, 0.2201 and 0.0597, respectively. The observed and expected phenotypes provide a good fit to Hardy-Weinberg equilibrium. The four alleles system will increase the probability of excluding a man falsely accused of paternity to 30% as compared with 16% if the two alleles system is used.

Étude du polymorphisme électrophorétique des lactate-, malate- et glutamate-déshydrogénases, de la phosphatase acide et des estérases de Providencia alcalifaciens, P. stuartii et P. rustigianii

The polymorphism of glutamate, lactate and malate dehydrogenases, of acid phosphatase and of esterases of 27 strains of Providencia alcalifaciens, 35 strains of P. stuartii and 17 strains of P. rustigianii was investigated by conventional electrophoresis in polyacrylamide agarose gel and by isoelectric focusing in thin-layer polyacrylamide gel. For each enzyme analysed, the three species were characterized by a distinct electrophoretic pattern. The number of allozymes detected by conventional electrophoresis was greater than that detected by isoelectric focusing. The use of these two techniques in parallel led to improved detection of polymorphism of esterase alpha beta from P. alcalifaciens. A two-dimensional profile obtained by plotting isoelectric points against electrophoretic mobilities for malate dehydrogenase, acid phosphatase and beta-A esterase showed a molecular relationship between the diverse allozymes and demonstrated their taxonomic values. Polymorphism varied considerably according to the enzyme and species analysed and was correlated with DNA heterogeneity. The strains of P. alcalifaciens exhibited the greatest enzyme polymorphism and were classified into two main zymotypes reflecting genetic divergence within this species, whereas the strains of P. stuartii were electrophoretically less variable.

Resolution of the isoenzymes of soybean lipoxygenase using isoelectric focusing and chromatofocusing

Isoelectric focusing in thin-layer polyacrylamide gels has been applied to the analysis of the enzymes involved in the formation and destruction of peroxides in soybeans [ Glycine max (L.)], lipoxygenases and peroxidases, respectively. As a result of differences in pH optima for catalytic activity, lipoxygenases were selectively detected by adjusting the pH employed for activity-specific staining. Type-1 lipoxygenase was revealed not only by staining based on the conversion of linoleic acid to hydroperoxide but also by two stains based on the reduction of the hydroperoxide. These methods were found to be suitable for the analysis and characterization of isoenzyme patterns in different soybean cultivars. A substantial difference in the distribution of lipoxygenases maximally active near pH 7 was observed for cultivars Provar and Vickery. A similar degree of separation of the isoenzymes was achieved on a larger scale using chromatofocusing in the pH range 7.4–5.0.

An immunoblotting technique for complement C6 typing: three new variants.

An immunoblotting method for C6 typing was developed. After isoelectric focusing in thin-layer polyacrylamide gel, C6 proteins were transferred passively to nitrocellulose and then detected by a two-step enzyme immunoassay. A population sample of northeastern Japanese was investigated using this method. Three common and four rare allotypes were observed. The allele frequencies estimated from 495 blood donors were as follows:C6*A0.423, C6*B0.510, C6*B20.062, and rare alleles (91,M11, B4, andB5) 0.005. Three variants, 91,M11, andB5, were considered to be newly found. This method could be applicable to many other protein systems.

Genetic variants of the human diaphorase DIA3 in Japanese: report of a new rare allele, DIA34.

Sperm-lysates from 264 unrelated Japanese males were tested for the polymorphism of the human diaphorase DIA3 by isoelectric focusing in thin-layer polyacrylamide gel over pH range 3.5–9.5 gradient. The occurrence of a new rare allele, DIA34, in addition to the previously described three alleles in German and British populations; has been demonstrated in our population. The estimated allele frequencies of the commonly encountered phenotypes,i.e. DIA31–1, 2–1, 3–1, 2–2 and 3–2, were: DIA31=0.837, DIA32=0.143 and DIA33=0.020.

Haemoglobin Hijiyama: a haemoglobin variant found in connection with glycosylated haemoglobin estimation in a Finnish diabetic boy.

A variant of haemoglobin A, named earlier haemoglobin Hijiyama, was found in three generations of a Finnish family in connection with the determination of glycosylated haemoglobin of a diabetic patient. The biochemical abnormality is in the beta-chain at residue 120 where lysine is replaced by glutamic acid. In the heterozygote carriers of the abnormal haemoglobin there was no apparent association with clinical or haematological abnormalities. By isoelectric focusing in thin-layer polyacrylamide gels, haemoglobin Hijiyama could be distinguished with high specificity from haemoglobin A as well as from other haemoglobin variants earlier found in Finland. The glycosylated haemoglobin fractions (haemoglobin A1 and haemoglobin Hijiyama 1) in the diabetic patient sample could be determined by colorimetric and electrophoretic methods.

Transferrin (Tf) polymorphism: An analysis by isoelectric focusing

The polymorphism of the transferrin (Tf) system was studied in a total of 300 unrelated Japanese individuals from Miyagi prefecture, the northern part of Japan, using isoelectric focusing in thin-layer polyacrylamide gel. In our population samples three common phenotypes and nine variants were observed. The calculated allele frequencies were Tf C 1 = 0.773, Tf C 2 = 0.212, Tf D chi (Chinese) = 0.008, and the combined frequencies for the Tf B var (variants) = 0.007. Family data ( n = 44) were in accordance with an autosomal codominant fashion of inheritance. The use of isoelectric focusing procedure among Japanese will raise the probability of excluding a man falsely accused of paternity to 15.8% as compared with 1.5% when the conventional electrophoretic methods are used.

Alpha 1-antitrypsin (pi) phenotypes in a village population from The Gambia, West Africa. Evidence of a new variant occurring at a polymorphic frequency.

A total of 701 individuals from a village in The Gambia, West Africa were tested for serum alpha 1-antitrypsin phenotypes by isoelectric focusing in thinlayer polyacrylamide gels (pH 4--5). A new variant allele, PiGAM, was discovered at a polymorphic frequency (0.0642), and the inheritance of the variant phenotype was confirmed by family studies. The variant was not found to be associated with any decrease in serum alpha 1-antitrypsin concentration. The only other allele found within this population was the common allele PiM (0.9358).

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gel: Application to cationic proteins in serum

Two-stage methods for alkaline-range analytical isoelectric focusing in thin-layer polyacrylamide gels have been developed for the analysis of the cationic proteins in complex mixtures of protein. A pH gradient covering the “entire” pH range but emphasizing only a narrow portion of this gradient is produced. During the first stage a nonlinear pH gradient is nearly established. The anodic electrode is then moved to a position on the gel such that the applied potential is exclusively across the alkaline portion of the gradient. The second stage results in a significant increase in the electric field strength and, consequently, the resolving power. The two-stage methods, exploiting the advantages of narrow pH gradients and high field strengths, have enabled excellent resolution of the highly heterogeneous cationic proteins in serum.

Isoelectric Focusing Studies on the -Fructofuranosidases and -Glucosidases of Streptococcus mitis

Extracts of Streptococcus mitis ATCC 903 were analysed for beta-fructofuranosidase and alpha-glucosidase activities by isoelectric focusing in thin-layer polyacrylamide gels combined with zymogram procedures. Three bands of activity were visualized in the gels after incubation with sucrose (pI 4.05, 4.25 and 4.85) and three other bands after incubation with p-nitrophenyl alpha-D-glucopyranoside (pI 3.90, 4.45 and 4.65). The enzymes responsible for the reaction with sucrose were identified as beta-fructofuranosidases (EC 3.2.1.26) for the following reasons: identical enzyme bands were visualized in the gels after incubation with raffinose; no enzyme bands appeared in the gel after incubation with the alpha-glucosides maltose, turanose, trehalose and melezitose; and the soluble fraction hydrolysed sucrose to equimolar amounts of glucose and fructose.

Immunofixation after electrofocusing: Improved method for specific detection of serum proteins with determination of isoelectric points I. Immunofixation print technique for detection of alpha-1-protease inhibitor

An improved method is described for direct localization of human serum proteins in polyacrylamide gel with simultaneous determination of their isoelectric points (pI). The technique employs isoelectric focusing in thin-layer polyacrylamide gels to separate the serum proteins and the pH gradient is read at 4°C with a dual-membrane surface microelectrode. Subsequently, the desired proteins are localized by immunofixation in the gel or by immunofixation-printing onto cellulose acetate strips soaked in specific antiserum. No sectioning of the electrofocused gel is necessary, and the entire technique can be completed in less than 14 h. When this method is applied to the detection of the genetic variants of alpha-1-antitrypsin (alpha-1-protease inhibitor) (A 1Pi system), the results indicate that it can be used to specifically localize serum proteins whose pI's differ by as little as 0.01 pH units. The resolution afforded is especially evident in the analysis of A 1Pi M variants.

Isoelectric focusing of milk-clotting enzymes

SummaryIsoelectric focusing in thin-layer polyacrylamide gel has been applied successfully to the characterization and identification of calf rennet and its substitutes. The use of acidic pH gradients (pH range 2·5–6) allows the identification of calf and microbial rennets and bovine and pig pepsins.A new, very rapid and sensitive zymogram technique has been developed by overlaying the gel with a highly porous, transparent cellulose acetate film impregnated with casein.

Protein profiles as an aid to taxonomy in the genus Diphyllobothrium.

Specific and distinct protein profiles were demonstrated by isoelectric focusing performed on soluble whole-body proteins of four Diphyllobothrium species. The protein patterns differed in number, position and relative density of the fractions separated. Altogether 31-36 protein bands per species were separated by isoelectric focusing in thin-layer polyacrylamide gels. The results show that sensitive chemotaxonomic methods can be used to help characterize and delimit species in the troublesome Diphyllobothrium group.