Focusing In Polyacrylamide Gels Research Articles

Carbohydrate-deficient glycoprotein syndrome: electrophoretic study of multiple serum glycoproteins

The serum glycoproteins in three patients with the carbohydrate-deficient glycoprotein (CDG) syndrome were examined using isoelectric focusing in polyacrylamide gels, SDS-PAGE, and a two-dimensional electrophoretic glycoproteins tested. Abnormal components of transferrin, antithrombin III and orosumucoid had smaller molecular weights, about 3000 and 6000 Da, than normal ones. Antithrombin III gave an increased cathodal band. Its pI and molecular weight were the same as those of a physiological variant of antithrombin III (β fraction), which is known to lack the asparagine-N-linked oligosaccharide chain at asparagine-135. The evidence suggests that a deficiency of asparagine-N-linked oligosaccharide transfer occurs in multiple serum glycoproteins in patients with CDG syndrome. Thus, examination of serum by isoelectric focusing in polyacrylamide gels and SDS-PAGE should be very useful for differentiating the CDG syndrome type I from clinical and biochemical variants.

Detection of pectinesterase in polyacrylamide gels.

A rapid and sensitive method of detecting pectinesterase activity following electrophoresis or isoelectric focusing in polyacrylamide gels is described. The method uses ruthenium red and requires no addition of substrate when making the gels, thus obviating direct enzyme-substrate contact during electrophoresis. Because of its versatility, the method can be used in a wide variety of applications, such as plant and microbial taxonomy, enzyme purification and characterization, or as an analytical method in fresh and processed plant technology.

Subtyping of coagulation factor XIIIA.

An extended polymorphism of the coagulation factor XIIIA can routinely be detected in human plasma samples and white cell lysates by isoelectric focusing in polyacrylamide gels containing 3 M urea in the pH range 5-8. Analyses of 184 families with 513 children confirmed the formal model proposed by Suzuki et al. [Am J Hum Genet 1988;43:170-174]. Four common alleles, F XIIIA*1A, 1B, 2A, 2B, at an autosomal locus control the expression of ten phenotypes. On the basis of the population sample from southwest Germany the frequencies of the common alleles F XIIIA*1A, 1B, 2A, 2B were calculated as 0.175, 0.609, 0.011, and 0.205, respectively.

Haemoglobin variants within the genus Cervus

Haemoglobins from a total of 168 red deer ( Cervus elaphus), five Japanese Sika deer ( Cervus nippon nippon), 49 hybrids between red deer and Sika deer ( Cervus elaphus × Cervus nippon) as well as 32 fallow deer ( Cervus dama) have been studied by isoelectric focusing in polyacrylamide gels. One single band showing no variation was found in all animals. All fallow deer exhibited an additional band. With respect to previous investigations on haemoglobin polymorphism, we must assume a high degree of genetic differentiation between European red deer populations at least for one haemoglobin gene locus. Gene duplication in the distant past or persistence of foetal haemoglobin are alternative hypotheses explaining the haemoglobin pattern of fallow deer.

A new αS1‐casein allele in bovine milk and its occurrence in different breeds

A new alpha S1-casein variant (alpha S1-CN F) with a frequency of 0.009 was demonstrated in a genetic resource of German Black and White Cattle by isoelectric focusing in polyacrylamide gels and by polyacrylamide gel electrophoresis under alkaline conditions. alpha S1-CN F was not present either in German Holstein Friesians, which originate from crossing Holstein Friesians with German Black and White Cattle, or in milk samples from eight other breeds in Germany.

The M20 IL-1 inhibitor: I. Purification by preparative isoelectric focusing in free solution

An interleukin-1 (IL-1) inhibitor produced by the M20 myelomonocytic cell line has been shown to be active in various in vitro and in vivo IL-1 induced parameters. This inhibitor has been purified from the conditioned medium by gel filtration through a Sephacryl S-300 column or dye ligand chromotography on Affi-Gel blue column, followed by isoelectric focusing in free solution in the pH range 3–5 using the Rotofor cell. When gel filtration by FPLC with the Superose 12 column was used as the final step, the combined sequence of purification procedures resulted in a 1600-fold purification of the IL-1 inhibitor. The purified IL-1 inhibitor has a molecular weight of approximately 52±4 kDa and a p I of 4.15±0.1. By SDS-PAGE analysis the inhibitor preparation thus obtained showed the presence of two protein bands, while a few closely spaced protein bands were seen by analytical isoelectric focusing in polyacrylamide gels (pH 3–6). Some of these bands in PAGIF might correspond to different degrees of glycosylation of the inhibitory protein. Although the M20 IL-1 inhibitor has not yet been purified to homogeneity, it should be stressed that the procedures used, allowed us to remove the great majority of the proteins present in the medium in which the M20 cells were cultured, and to recover in satisfactory yield the inhibitor which we consider likely to be present in the conditioned medium in subnanomolar concentrations.

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.

Microdetermination of G6PD isoenzyme activity in human erythrocytes by thin-layer PAG-IEF

An improved method of microdetermination of G6PD isoenzyme activity in human erythrocytes was developed by modification of previously reported procedures. The volume of blood samples was reduced from 2 ml to 20 μl. After hemolysis in 2% Triton X-100 and 0.1% β-mercaptoethanol, the samples were subjected to centrifugation and thin-layer isoelectric focusing in polyacrylamide gel (PAG-IEF). By comparison with the original method, excellent resolution was obtained by this more rapid and simple procedure.

Purification and Properties of Steroid Sulfatase from Human Placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.

Genetic and biochemical characterization of a new chymotrypsin isozyme of the house mouse, CTRA-1

Genetic variation of a codominantly inherited pancreas protease, designated CTRA-1, was discovered in the house mouse by isoelectric focusing in polyacrylamide gels. Phenotype CTRA-1A was found in MOLH/Fre and in the majority of common laboratory mouse strains. Phenotype CTRA-1B was found in PWD/Ph. It was characterized by the absence of a corresponding protease band. A third phenotype, CTRA-1C, was observed in IS/Cam and a fourth phenotype, CTRA-1D, was detected in SEG/1. CTRA-1 was found only in the pancreas and may represent the A form of chymotrypsin. The enzyme was shown to be controlled by the presumed structural locus Ctra-1 located on chromosome 8. From two backcross series, including a total of 274 animals, the gene order (Es-1, Es-9)-3.9 +/- 1.7%-Got-2-3.9 +/- 1.7%-(Es-2, Es-7, Es-23)-0.7 +/- 0.5%- Ctra-1-6.3 +/- 2.2%-Prt-2 was established.

Use of enzyme electrophoresis for differentiating Schistosoma nasale and S. spindale infections of Indoplanorbis exustus in Sri Lanka

Indoplanorbis exustus is the intermediate snail host of both Schistosoma nasale and S. spindale in Sri Lanka. Due to the similar morphology of the cercariae difficulties exist in identifying the species of schistosome infecting snails at transmission sites. Isoelectric focusing in polyacrylamide gels of malate dehydrogenase, glucose phosphate isomerase, phosphoglucomutase and acid phosphatase has been carried out on extracts of cercariae and potentially useful diagnostic markers have been identified. Cellulose acetate membrane electrophoresis has been used to separate two glucose phosphate isomerase phenotypes each of which appears to be diagnostic for one of the species. This simple electrophoretic technique has been used to identify schistosome infections in Indoplanorbis exustus collected at two transmission sites in Sri Lanka. Out of a total of 1,240 snails collected, 13 snails were shown to be infected with S. nasale, 19 with S. spindale and two snails were found to have mixed infections.

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate: sugar phosphotransferase system in streptococci

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (p I = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100°C for 20 min or storage at −40°C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested ( S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis. A single form was detected in L casei, E faecalis, S aureus, and B subtilis. It thus appears that the presence of 2 forms of HPr is restricted to the genera Streptococcus and Lactococcus.

Use of a fast protein electrophoretic purification procedure for N‐terminal sequence analysis to identify S‐locus related proteins in stigmas of Brassica oleracea

In the cruciferous plant Brassica oleracea L. (cabbage), the S-locus specific glycoproteins (SLSGs) isolated only in stigmas are considered to play an important role in the normal prevention of self-fertilization. Recent molecular data have shown that the gene encoding these glycoproteins (the SLG gene) belonged to a multigenic family consisting of about 10 homologous copies among which another member is expressed, the S-locus related gene (SLR1gene). Our aim was to determine whether the SLR1-gene proteins were expressed in the stigmatic tissues. We first identified the putative SLSGs or SLR1-proteins by Con A-peroxidase detection of glycoproteins separated after isoelectric focusing in polyacrylamide gels. We describe a fast purification procedure for the glycoproteins of interest, based on analytical isoelectric focusing, electrophoresis, and electroblotting of proteins onto polyvinylidene difluoride membranes. Blotted proteins were sequenced for N-terminal amino acid determination. By comparison of the N-terminal sequences of the purified proteins with the peptide sequence predicted from the SLR1-cDNA, we demonstrate the expression of SLR1-like proteins in stigmas of B. oleracea.

Polymorphism of the Plasminogen (PLG) System in Cádiz Province, Southern Spain

In this work, the authors report a plasminogen (PLG) system genetic-population study in a sample of 378 healthy subjects, of both sexes and unrelated, all resident in the province of Cádiz in Southern Spain. In this study, the PLG types were determined by isoelectric focusing in polyacrylamide gels (PAGIF), followed by staining with Coomassie blue. The genic frequencies obtained were the following: PLG A = 0.833 333 3; PLG B = 0.166 666 7.

Characterization of membrane-associated arginine aminopeptidase inStreptococcus sanguis 903

Extracts of cytoplasmic membranes ofStreptococcus sanguis 903 were analyzed for aminopeptidase activity by isoelectric focusing in polyacrylamide gel and enzyme staining with 16 different aminopeptidase substrates. A single aminopeptidase with specificity for aminoterminal arginine was detected. The enzyme was stimulated by dithiothreitol andβ-mercaptoethanol. Urea, sodium dodecyl sulfate (SDS), andp-chloromercuribenzoate were inhibitory. Metal ions had little or no effect on activity, except that Hg2+, Cu2+, and Ni2+ were inhibitory. The pH optimum for activity was at 7.2. The molecular mass estimated by SDS-polyacrylamide gel electrophoresis was 170 kDa.

Observations on natural and experimental interactions between Schistosoma bovis and S. curassoni from West Africa

Surveys of 332 naturally infected bovines at eight abattoirs in Senegal, The Gambia and Mali were carried out to determine the prevalence of infection with Schistosoma bovis and S. curassoni and to pinpoint areas where the distribution of the species overlap. S. bovis was the commonest schistosome of cattle in Senegal and Mali being found in animals at seven abattoirs, the highest prevalance of 85.1% occurred at Mopti in Mali. S. bovis was the only bovine schistosome observed in The Gambia. S. curassoni was isolated from a cow at Bamako and shown to have similar glucose-6-phosphate dehydrogenase, phosphoglucomutase and acid phosphatase profiles to those described for a Senegalese isolate. Evidence of interaction of S. bovis with S. curassoni was found in cattle from Senegal, at Tambacounda and Kolda, and from Mali, at Bamako and Mopti. A mixed experimental infection of both species in a sheep showed the lack of any specific mate recognition system: identification of the worms was facilitated by analysis of acid phosphatase by isoelectric focusing in polyacrylamide gels. Viable hybrid parasites were produced in the laboratory and were maintained up until the F 4 generation. Comparisons of egg morphology, surface structure of adult male worms and enzyme profiles have been made between experimental hybrid lines and field isolates. Possible mechanisms maintaining species integrity are discussed.

Purification of a novel insulin-stimulated protein kinase from rat liver.

We previously described a novel insulin-stimulated protein kinase activity that phosphorylates Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) in cytosolic extracts of adipocytes (Yu, K-T., Khalaf, N., and Czech, M. P. (1987) J. Biol. Chem. 262, 16677-16685). In the present experiments, cytosolic extracts of livers from insulin-treated rats also exhibited a 30-100% increase in this Kemptide kinase activity and served as an abundant source for purification. The Kemptide kinase was purified in parallel from liver extracts of insulin-treated or control rats through five chromatographic steps and one polyethylene glycol precipitation. The chromatographic behavior of the insulin-stimulated Kemptide kinase differed significantly from the control kinase on Mono Q and heparin-Sepharose resins. The purified kinase preparations retain insulin stimulations of 2-10-fold. Analysis of the purified control and insulin-stimulated kinases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed single bands with similar silver staining intensity and apparent molecular masses of 52 kDa. The insulin-stimulated Kemptide phosphorylating activity also coincided with the major silver-stained band following isoelectric focusing in polyacrylamide gels. The stimulation of kinase activity in response to administration of insulin is due to an increase in Vmax, whereas the Km for Kemptide (0.3 mM) is unchanged. The apparent molecular mass of the native kinase determined by gel filtration is approximately 50 kDa, suggesting that it exists as a monomer. Either Mg2+ or Mn2+ serve as cofactors for the kinase which phosphorylates a variety of basic substrates including a number of peptides and histones. The activity of the Kemptide kinase is not changed by several compounds that have been shown to modulate other kinases. Based on these data, we conclude 1) a novel insulin-sensitive Kemptide kinase in liver cytosol has been purified to near homogeneity, and 2) insulin administration acutely modulates the specific activity of this Kemptide kinase in livers of intact rats.

Comparison of isoenzyme profiles ofToxoplasma gondii tachyzoites produced under different culture conditions

The isoenzyme profiles for ten enzymes of tachyzoites either grown in vivo in TG 180 mouse sarcoma cells or cultivated in vitro in human fibroblast cell culture were compared in eight cloned strains by means of isoelectric focusing in polyacrylamide gels. Fibroblast-cultivated tachyzoites of all strains tested showed an extra band in the LDH isoenzyme pattern that was not seen in tachyzoites from TG 180 mouse sarcoma cells. The isoenzyme patterns of four enzymes [aspartate aminotransferase (ASAT), glutathione reductase (GSR), glucose phosphate isomerase (GPI) and amylase] used to differentiate the strains did not change under the two different culture conditions. Nevertheless, the shift observed in the LDH isoenzyme pattern underlines the fact that isoenzyme analysis of Toxoplasma strains could be carried out with tachyzoites produced under the same culture conditions.

Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient

The production of anodic, cathodic and symmetrical drifts of a pH 3.5-10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3-10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.

Monocyte‐specific esterase isoenzyme demonstrated by isoelectric focusing

Using horizontal thin-layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2-11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI 6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono-bands. Dilution experiments established the distinct prominence of the mono-band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono-band negative leukemic cells whose arrest of differentiation can be overcome by in vitro 12-O-tetradecanoylphorbol 13-acetate-promoted differentiation to more mature cells, could be induced to express the mono-band which paralleled their maturation to monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)