Cellogel Electrophoresis Research Articles

Primary structure of bovine lactate dehydrogenase-A isozyme and its synthesis in Escherichia coli

Eleven cDNA clones encoding lactate dehydrogenase(LDH)-A isozyme were isolated from a bovine lymphocyte cDNA library, and the nucleotide sequences of three of the clones (pLDH5, pLDH9 and pLDH12) were determined. With the exception of variation in the 5′ portion, two cDNA clones (pLDH9 and pLDH12) appeared to contain the full-length cDNA of 1786 bp, consisting of the protein-coding sequence (996 bp), the 5′- and the 3′-untranslated regions and the poly(dA) tail. The predicted amino acid (aa) sequence of bovine LDH-A (332 aa) showed 96.7% homology with that of pig LDH-A. The protein-coding cDNA region (1650 bp) was inserted into an Escherichia coli expression vector ptac11 and expressed. The protein synthesized E. coli showed enzyme activity of LDH and was identified by cellogel electrophoresis as LDH-5 isozyme whose subunit M chain is the product of the LDH-A gene.

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P 2) an effector of the delivery of O 2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The biphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

A cladistic analysis of species of the molluscan genusPotamopyrgusbased on allozyme data

The relationships among four species of the molluscan genus Potamopyrgus have been investigated using cellogel electrophoresis. Electrophoretic variability at nine presumptive loci was calculated. Cladograms were constructed using both electromorph and locus as characters. Results indicate two major groupings within the genus. P. estuarinus and P. pupoides appear to represent sister taxa and, as a grouping, are closely related to P. antipodarum. The relationship of P. troglodytes to other species of the genus remains problematical. The cladograms obtained are congruent with that based on morphology with respecttoP. antipodarum, P. estuarinus, and P. pupoides.

Systematics and Mitochondrial Genome Evolution of Australian Rosellas (Aves: Platycercidae)

.We used six restriction endonucleases (D&I, HinfI, HpaII, MM, TagI, and Zmo37), each with a unique four-base recognition site, to digest 26 mitochondrial genomes from six Platycercus species in an investigation of the systematics of this speciose and variable Australian genus. The resulting fragments were end labeled and separated by means of polyacrylamide-gel electrophoresis. The number of substitutions per nucleotide site between each of the genomes was estimated on the basis of the distribution of fragments between samples by means of the method of Nei and Li. These pairwise distances were used to construct evolutionary trees either by assuming an evolutionary clock or by allowing the branch lengths to vary independently. The presence or absence of each restriction fragment was also scored and used as characters in Wagner parsimony analysis. In general, the phylogenies produced from mitochondrial genome diversity mirror the current classification of the genus, except for P. icterotis, which is not clustered with either of the superspecies of Platycercus and, as such, should probably be placed in a third group. To confirm that the family of parrots to which Platycercus belongs has a conservative nuclear genome, Nei’s genetic distance between and within Platycercus and other related species was estimated by means of cellogel electrophoresis of liver isozymes. Of the 26 Platycercus mitochondrial genomes studied, 23 different haplotypes were identified according to the limits of resolution of the technique. The mean interspecific mitochondrial genome diversity was 0.0457, and the intraspecific genome diversity varied between 0.003 1 (for P. caledonicus) and 0.0 142 (for P. eximius). These diversity values indicate either that the rate of rosella genome evolution is 2-3 orders of magnitude below the rate reported for primates or that the effective sizes of the rosella populations are 2-3 orders of magnitude below the sizes estimated on the basis of ecological methods.

G6PD Punjab, a dialysis sensitive variant of human glucose-6-phosphate dehydrogenase

Erythrocyte samples from 101 individuals, originally from Punjab and living at the time of investigation in England, were screened for glucose-6-phosphate dehydrogenase (G6PD) variants by Beutler’s fluorescent spot test and standard cellulose acetate gel (Cellogel) electrophoresis. All but 2 of the 40 males in the study were found to be indistinguishable from normal G6PD B. One of the variants had 2% of the normal activity and resembled G6PD Mediterranean in electrophoretic behaviour. The other variant showed 52% of the normal activity and migrated slower than G6PD B in Cellogel with about half of the normal band intensity. A set of physicochemical characteristics of the variant determined by conventional methods distinguished it from the variants reported so far. It was designated as G6PD Punjab, and the corresponding allele asG6PD PUN. The most striking feature of G6PD Punjab is a remarkable alteration in its electrophoretic behaviour after dialysis.

Electrophoretic study of some enzymatic genes inMusca domesiicaL.

Abstract Cellogel electrophoresis has been used to study isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), malic enzyme (ME), and alcohol dehydrogenase (ADH), in the housefly. Two forms of MDH (MDH, and MDH2) have been separated. These show properties similar to those ascribed to supernatant and mitochondrial MDH enzymes in other organisms. In the housefly zymograms of the mitochondrial fraction show MDH2 activity only. Allelic variants of MDH1 differing in thermostability and resistance to p‐hydroxymercuribenzoate have been revealed by qualitative analysis. MDH2 is invariant. These findings indicate that the two forms are isozymes under independent genetic control. Other enzymes examined here exhibit only one isozyme, which shows genetic polymorphism in some strains. Using the variants and visible markers, the linkage groups of the structural loci of the enzymes have been identified: for some of them the genetic map position has been determined.

Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus

The expression of maternally derived X-chromosomal Pgk-1 alleles was investigated in oocytes and early embryos of mice carrying different alleles ( Xce a, Xce c) of the X-chromosome controlling element ( Xce) locus. Pgk-1 allelic expression was determined by measuring their gene products using Cellogel electrophoresis and a sensitive fluorimetric enzyme assay. In addition to the already existing mouse strain of the genotypes Pgk-1 a Xce c and Pgk-1 b Xce a, a new line was bred carrying the combination Pgk-1 b Xce c. The × chromosomes carrying the combinations Pgk-1 a Xce c and Pgk-1 b Xce c were of feral origin, whereas Pgk-1 b Xce a was derived from a laboratory line. Our results using Xce c homozygous females confirm that maternal Pgk-1 is already expressed on day 4 of embryogenesis, thus substantiating data previously obtained using Xce a/ Xce c heterozygous females. This finding also demonstrates that the timing of reactivation of maternal Pgk-1 is not influenced by the Xce locus. Furthermore, we found that oocytes from Xce c homozygous females have a balanced PGK-1A/PGK-1B allozyme ratio (50:50), whereas in oocytes obtained from Xce a/ Xce c heterozygotes, the PGK-1 allozyme ratio is about 60:40. In tissues of adult Xce homozygous females, the PGK-1 allozymes are also balanced, whereas in Xce a/ Xce c heterozygous females, the ratio is about 35:65. In addition to the relative activity of the PGK-1 allozymes, we also measured the absolute activity of PGK-1 in oocytes obtained from three types of Xce homozygous females. Oocytes from Xce c homozygous females were shown to have only half of the PGK-1 activity (independent of the Pgk-1 allele linked to Xce c) exhibited by oocytes obtained from Xce a homozygotes. In contrast, the activity of G6PD, another X-linked enzyme, is not influenced by the Xce locus.

Gene mapping in chicken-Chinese hamster somatic cell hybrids. Serum albumin and phosphoglucomutase-2 structural genes on chicken chromosome 6.

Chicken phosphoglucomutase (PGM-2), serum albumin, vitamin D binding protein (Gc) and phosphoribosyl pyrophosphate amidotransferase (PPAT) structural genes have been mapped to chicken chromosome 6 using chicken-Chinese hamster somatic cell hybrids containing this chromosome as the only chicken genetic material. Chicken PGM-2 activity was detected in the hybrids using cellogel electrophoresis and a substrate, ribose-1-phosphate (R-1-P), that allows the detection of PGM-1 activity in mice and PGM-2 activity in humans. Chicken albumin sequences were detected in the hybrids with the use of a labelled chicken serum albumin cloned cDNA. Classical studies have shown linkage of the serum albumin and Gc genes, and the Gc gene also can be localized to chicken chromosome 6. The PPAT gene was localized to this chromosome in previous studies using these hybrids. A homologous linkage group has been identified in mammals and, therefore, a chromosomal linkage group containing at least four genes--Gc, serum albumin, PPAT, and PGM-2--has been conserved over a period of 300 million years, throughout both avian and mammalian evolution.

Purification of the M-type phosphoglyceromutase from rabbit muscle

A new method for the purification of M-type phosphoglyceromutase from rabbit muscle involving affinity chromatography on dye ligand media in the presence of 2,3-diphosphoglycerate is described. The method is rapid and technically simple. The purity of the enzyme was verified by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate and by Cellogel electrophoresis. Immunological techniques showed that the anti-M antibodies are specific and do not cross-react with the B isozyme.

An immunochemical method for detecting an inactive enzyme (bisphosphoglyceromutase) after Cellogel electrophoresis

AbstractA new method is described for detecting inactive enzymes or other proteins after Cellogel electrophoresis. The proteins were blotted from the Cellogel strip to a nitro‐cellulose membrane and then the blot was blocked before incubation in a specific antibody solution. The antigen‐antibody complex was located by a second antibody staining procedure employing peroxidase‐linked goat anti‐rabbit IgG. The method described is sensitive and could be of general use for the detection of proteins after Cellogel electrophoresis.

Polymorphism of Red Cell Adenosine Deaminase by Cellogel Electrophoresis in Rabbits

Using cellogel electrophoresis, polymorphism of adenosine deaminase (ADA) was found in haemolysed rabbit erythrocytes. In this study, six different phenotypes A, B, C, AB, AC and BC were observed. It was confirmed from family data that these phenotypes were determined by three codominant alleles ADAA ADAB and ADAC.

A new simple blot transfer method following cellogel electrophoresis which preserves antibody activity: Application to the analysis of monoclonal and oligoclonal fluids

Electrophoresis on gelatinized cellulose acetate (cellogel), followed by a simple blot transfer onto a nitrocellulose membrane, allows analysis of the electrophoretic pattern of specific antibody-active molecules by their binding of radiolabelled antigen. Using this non-denaturing method, it is easy to establish the monoclonal or oligoclonal nature of an antibody activity present in a serum or to control the cloning of a hybridoma cell line. Comparison between protein staining and autoradiography can afford a rough estimation of the relative affinities of different monoclonal antibodies towards the same antigen (in this case, the hepatitis B surface antigen). In addition, inhibition studies using other monoclonal antibodies directed against the same antigen may help to delineate the epitope specificity of antibodies.

Simultaneous typing of C3 and Tf and of Gc and Bf on Cellogel electrophoresis

A method is described to type C3 and Tf on the same Cellogel strip (and Bf simultaneously on a parallel strip). A different method allows to type Gc and Bf on one Cellogel strip. These methods are rapid, easy and reliable, although agarose electrophoresis is a little more effective.

Diagnosis of Pseudo-Arylsulfatase A Deficiency with Electrophoretic Techniques

Summary: Deficient arylsulfatase A activity in man has long been associated with the neurodegenerative disease, metachromatic leukodystrophy. However, similar deficiency has been noted in clinically normal individuals, and is referred to as the pseudoarylsulfatase A deficiency condition. Although direct quantitative analysis of arylsulfatase A activity failed to differentiate between these two conditions, analysis of residual arylsulfatase A activity with either Cellogel electrophoresis or isoelectric focusing in polyacrylamide gels now has been shown to distinguish between them unequivocally. With both techniques, cultured fibroblasts from patients with pseudo-arylsulfatase A deficiency showed faint but clear bands of arylsulfatase A activity. Under identical conditions, fibroblasts from patients with metachromatic leukodystrophy showed no trace of activity. These methods can be adapted easily for general laboratory analysis in cases when results from quantitative arylsulfatase A assays are noninformative.

Diagnosis of pseudo-arylsulfatase A deficiency with electrophoretic techniques.

Deficient arylsulfatase A activity in man has long been associated with the neurodegenerative disease, metachromatic leukodystrophy. However, similar deficiency has been noted in clinically normal individuals, and is referred to as the pseudoarylsulfatase A deficiency condition. Although direct quantitative analysis of arylsulfatase A activity failed to differentiate between these two conditions, analysis of residual arylsulfatase A activity with either Cellogel electrophoresis or isoelectric focusing in polyacrylamide gels now has been shown to distinguish between them unequivocally. With both techniques, cultured fibroblasts from patients with pseudo-arylsulfatase A deficiency showed faint but clear bands of arylsulfatase A activity. Under identical conditions, fibroblasts from patients with metachromatic leukodystrophy showed no trace of activity. These methods can be adapted easily for general laboratory analysis in cases when results from quantitative arylsulfatase A assays are noninformative.

Glyoxalase I polymorphism in Belgium.

The phenotypes of red cell glyoxalase I (EC 4.4.1.5) were determined by cellogel electrophoresis in 750 Belgian persons. The gene frequencies found were 0.432 for GLO1 and 0.568 for GLO2.

Oestradiol induced changes of soluble protein synthesis in human endometrium

The effect of an in vitro application of oestradiol-17-β on the soluble protein synthesis in human endometrium during the menstrual cycle was investigated. Results obtained by double-label technique and SDS-polyacrylamide gel or Cellogel electrophoresis show that synthesis of soluble proteins in human endometrium is affected by oestradiol in both follicular and luteal phases of the cycle. One of the proteins obtained after oestradiol treatment seems to be similar to IP of rat uterus as judged from its molecular weight (41,000 daltons) and electrophoretic mobility R F : 1.18–1.24 relative to BSA on Cellogel). The magnitude of this protein synthesis correlates significantly with cytoplasmic oestradiol receptor concentration in endometrium throughout the cycle. The highest induction of IP like protein(s) synthesis at follicular phase and a decreased induction during the luteal phase was detected. No quantitative relationship between the changes of progesterone receptor concentration and IP like protein synthesis was found.

Studies of the Regan alkaline phosphatase isoenzyme in plasma from a lung-carcinoma patient.

We compared an alkaline phosphatase (EC 3.1.3.1) isoenzyme from a plasma sample of a patient with carcinoma of the lung, who was suspected of having the Regan-type isoenzyme, with placental serum alkaline phosphatase and with tissue preparations from placenta, liver, bone, and intestine. The placental and Regan phosphatases responded similarly to heat treatment and modification by various inhibitors, except that the Regan isoenzyme was more resistant to EDTA inactivation than was the placental serum alkaline phosphatase, but more sensitive than placental tissue alkaline phosphatase. Isoelectric focusing and Cellogel electrophoresis showed the Regan isoenzyme to have a mobility similar to that of the placental type. We conclude that the isoenzyme was of the Regan type and support the contention that the placental and Regan isoenzymes are products of the same structural gene, with small differences due to post-genetic modification.

Analysis of purine nucleoside phosphorylase from human T and non-T lymphocytes of different maturation stages

In the last few years evidence has been growing that purine/pyrimidine metabolism changes during maturation of lymphocytes. In this view enzyme characteristics of lymphocytes may act as biochemical markers of lymphocyte maturation. Human purine nucleoside phosphorylase (PNP) has been studied in extracts of T lymphocytes isolated from thymic tissue, cord blood, and peripheral blood, as well as in extracts of non-T lymphocytes from cord blood, peripheral blood and tonsils. Using inosine or deoxyinosine as a substrate, complicated kinetics of PNP are observed which may be caused by the presence of multiple active enzyme species. The number of PNP-active molecular forms increases with the maturation stage of the T and B lymphocytes as shown by cellogel electrophoresis. PNP activity of thymocytes as compared to peripheral blood T cells is low; PNP activity of peripheral blood B cells as compared to neonatal B cells does not differ significantly. The difference found in PNP activity between thymocytes and peripheral blood T cells is caused by the difference in amount of PNP protein, as has been demonstrated by immunoprecipitation tests.

Creatine kinase from the electric organ of Electrophorus electricus (L.)—isozyme analysis

1. 1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract. 2. 2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity. 3. 3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure. 4. 4. The implications of this finding with the muscular origin of the electric organ are discussed.