Electrophoretic Forms Research Articles

In Ovo Competition between Distinct Virus Populations in an Avian Influenza Isolate

Embryo lethality patterns induced by an avian influenza virus isolate (A/Tk/Ws/68/H5N9) suggested that it contained more than one genetic form. Two different virus populations were recovered from the isolate by plaque isolation and limit-dilution cloning and were characterized with respect to their biological and molecular properties. They were very closely related but exhibited strikingly different mean death times (MDT) in 10-day-old chick embryos. One was rapidly embryo lethal (REL), while the other was slowly embryo lethal (SEL). The REL isolate demonstrated a small but measurable mortality rate in 4-week-old chicks, as did the parental isolate. The SEL isolate, however, was nonlethal to 4-week-old chicks. The embryo MDT induced by the parental isolate revealed a biphasic death pattern reflecting expression of both REL and SEL populations. Mixing experiments, using different amounts of the two cloned populations, demonstrated that expression of their unique phenotypic property (either REL or SEL) was competitive. The number of early or late embryo deaths was directly related to the input levels of each respective virus. The only molecular difference thus far detected between the two populations is in the nonstructural (NS) gene, with the REL clone possessing a faster migrating electrophoretic form of that RNA than the SEL clone. Both forms of the NS gene were present in the original parental isolate. This study thus demonstrates the competitive coexistence of two closely related virus populations within a single natural isolate.

Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.

Immunoblotting experiments with polyclonal and monoclonal anti-transcription factor IIIA (TFIIIA) antibodies reveal different electrophoretic forms of TFIIIA in extracts from immature and mature oocytes of Xenopus laevis. The well-characterized 39-kD TFIIIA species is present in approximately 10(12) copies per cell in stage I-III previtellogenic oocytes and declines in abundance by 10- to 20-fold during oogenesis. An immunologically related protein of apparent molecular mass of 42 kD is present at 2-4% of the level of 39-kD TFIIIA in immature oocytes, and the level of this protein increases dramatically during oogenesis. Both the 39- and 42-kD proteins are complexed with 5S RNA in 7S ribonucleoprotein (RNP) particles. High-level transcription of the oocyte-type 5S genes in vitro requires 39-kD immature oocyte TFIIIA, whereas both 39-kD TFIIIA and the mature oocyte TFIIIA species of 42 kD support somatic-type 5S transcription. TFIIIA of 42 kD does not support oocyte-type 5S transcription in a fractionated transcription system derived from mature oocytes. Both proteins, however, bind the oocyte-type and somatic-type genes with comparable affinities and exhibit similar DNase footprints on both genes. These results suggest a model for the developmental regulation of 5S RNA gene transcription where 42-kD TFIIIA serves as an activator of somatic-type 5S transcription and as a repressor of oocyte-type transcription during early embryogenesis.

Regulated phosphorylation and dephosphorylation of GAL4, a transcriptional activator.

In yeast, galactose triggers a rapid GAL4-dependent induction of galactose/melibiose regulon (GAL/MEL) gene transcription, and glucose represses this activation. We discovered that alterations in the physical state of the GAL4 protein correlate with activation and repression of the GAL/MEL genes. Using Western immunoblot assay, we observe two electrophoretic forms of GAL4 protein-GAL4I and GAL4II-in noninduced cells. In the absence of glucose, the addition of galactose to such cells results in the rapid appearance of a third and slower-migrating form, GAL4III, which differs from at least GAL4I by phosphorylation. GAL80-deletion cells that constitutively transcribe galactose-responsive genes due to the lack of the GAL80 protein, an antagonist of the GAL4 protein, exhibit GAL4III without galactose addition. Addition of glucose, which results in rapid repression of galactose gene transcription, triggers a rapid elimination of GAL4III and an increase in GAL4II. Cycloheximide experiments provide evidence that the galactose- and glucose-triggered GAL4 protein mobility shifts are due to post-translational modification. GAL4III is labeled with [32P]phosphate in vivo; in vivo 35S-labeled GAL4III could be converted by phosphatase treatment in vitro to GAL4I. We present a model proposing that phosphorylation state changes in the GAL4 protein are key to modulating its activity.

Plasmodium vivax: enzyme polymorphism in isolates of Indian origin

185 isolates of Plasmodium vivax were collected from patients visiting the malaria clinic run by the National Malaria Eradication Programme, Delhi, India. Percoll gradient centrifugation was used to concentrate P. vivax parasites from 0·4 to 0·5 ml of blood collected by finger prick. The parasite concentrate from each isolate was electrophoretically analysed for lactate dehydrogenase (LDH), NADP-dependent glutamate dehydrogenase (GDH), glucose phosphate isomerase (GPI) and adenosine deaminase (ADA). Variations were observed in GPI, GDH and ADA systems. Four electrophoretic forms of GPI and 5 each of GDH and ADA were observed. Electrophoretic mobilities of the different isoenzymic forms in P. vivax were identical to those reported for P. falciparum, indicating that the 2 species cannot be differentiated on the basis of electrophoretic patterns of the 4 enzyme systems studied.

Enzyme variation and pathogenicity of recent field isolates of Eimeria tenella

Seventy isolates of Eimeria tenella, obtained from commercial poultry farms worldwide and four reference laboratory strains were characterised by studies on the electrophoretic mobility of up to three enzymes. All populations possessed the same electrophoretic form of lactate dehydrogenase and malate dehydrogenase and one of two forms of glucose phosphate isomerase. One isolate was characterised by both forms of glucose phosphate isomerase. Studies on several isolates indicated that there was no correlation between the form of glucose phosphate isomerase found and the pathogenicity of an isolate.

Antigenic variation in alkaline deoxyribonuclease induced by three different strains of Epstein‐Barr virus

To determine whether biological and/or biochemical variants exist between strains of Epstein-Barr virus (EBV), we superinfected Raji cells with the nontransforming lytic strain of EBV (HR-1), and two isolates that both transform B-lymphocytes and superinfect Raji cells, B95-8, and NPC-EBV. The superinfected cells were assayed for EBV specific DNase. A new electrophoretic form of DNase was observed in cells superinfected with B95-8 EBV as compared to the enzymes induced by the HR-1 and NPC-EBV isolates. There were antigenic differences in the DNase induced by the EBV strains. Since antibody to EBV DNase is a marker for nasopharyngeal carcinoma (NPC), these data may have implications for EBV-associated disease.

Characterization of a human cytomegalovirus glycoprotein complex (gcI).

Three distinct families of glycoprotein complexes present in the envelopes of human cytomegalovirus and designated gcI, gcII and gcIII have been described recently. The synthesis of the gcI family was analysed using either inhibitors of glycoprotein processing and transport or endoglycosidase treatments of purified glycoproteins. The initial step in gcI synthesis involved the glycosylation of a 95K protein (p95) to form a high-mannose, simple N-linked glycoprotein of Mr 158K (gp158), which was detected only in the presence of the glycoprotein processing inhibitor castanospermine. This intermediate was rapidly trimmed in the virus-infected cell to form a more stable simple N-linked precursor glycoprotein of Mr 138K (gp138). Treatment of either gp158 or gp138 with endoglycosidase H produced p95. Both molecules, gp158 and gp138, were found in disulphide-linked complexes which are presumably infected cell precursors to gcI since they were not found in virions. The processing of these complexes involved complete cleavage of gp138 and conversion of some but not all of its oligosaccharide to complex N-linked chains. Both processing events were inhibited by the ionophore monensin. Mature gcI contained the gp138 cleavage product, gp93-130. The latter glycoprotein could be separated into two electrophoretic forms, gp93 and gp130. The deglycosylated form of gp55 had a discrete banding pattern with an apparent Mr of 46K (p46). In contrast, the deglycosylated forms of gp93 and gp130 had diffuse banding patterns with apparent Mr values of 46K to 56K (p46-56) and 60K to 70K (p60-70) respectively. Peptide profiles comparing gp93 with gp130 indicated that they have highly similar polypeptide backbones. Since the deglycosylated forms of gp55 and gp130, 46K and 60K to 70K, respectively, together exceed the 95K precursor/deglycosylated intermediate in Mr, we propose that the above glycoproteins are derived by an alternative proteolytic cleavage of the precursor. The heterogeneous electrophoretic properties of the deglycosylated forms of gp93 and gp130 may be due to additional post-translational modifications other than glycosylation.

Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus

Several disulfide-linked glycoprotein complexes were identified in the envelope of human cytomegalovirus (HCMV). These glycoprotein complexes were fractionated by rate-zonal centrifugation in sucrose density gradients in the presence of detergents. Fractionated glycoproteins and complexes were immunoprecipitated with three different monoclonal antibodies specific for HCMV glycoproteins and a rabbit polyclonal antiserum prepared against detergent-extracted virion and dense-body envelope glycoproteins. Three distinct families of disulfide-linked glycoprotein complexes were observed and designated glycoprotein complex gcI, gcII, and gcIII. The gcI family, recognized by monoclonal antibody 41C2 under nonreducing conditions, consisted of three complexes with approximate molecular masses of 250 to 300, 190, and 160 kilodaltons (kDa). These complexes consistently sediment more rapidly than other HCMV glycoproteins or complexes in sucrose density gradients. Upon reduction of the gcI family, two size classes of glycoproteins with average molecular masses of 93 to 130 and 55 kDa were observed. The gcII family was recognized by monoclonal antibody 9E10. Under nonreducing conditions, as many as six electrophoretic forms were observed for gcII. When reduced, the major component of the gcII family was a heterogeneous glycoprotein designated gp47-52. The gcIII family was recognized by monoclonal antibody 1G6. It consisted of a complex of approximately 240 kDa without reduction of disulfide bonds. When reduced, two glycoprotein size classes with average molecular masses of 145 and 86 kDa were observed. Polyclonal antiserum R-7 reacted strongly with the gcI and gcIII families, but weakly with the gcII family.

Two-dimensional electrophoretic analysis of human Cu,Zn-superoxide dismutase.

Three electrophoretic techniques, isoelectric focusing, polyacrylamide electrophoresis and sodium dodecyl sulfate-polyacrylamide electrophoresis, were combined to develop a projective two-dimensional electrophoretic technique. The new technique was applied to analysis of human Cu, Zn-superoxide dismutase (SOD) isozymes. By using isoelectric focusing, human Cu, Zn-SOD resolved into five isozymes, which are thought to be charge isomers. From the result of the two-dimensional electrophoresis without denaturing agents, each isozyme was found to possess two or three electrophoretic forms, which were supposed to be in equilibrium. The five bands separated by electrophoresis thus contain several electrophoretic forms of SOD isozymes. The projective two-dimensional electrophoretic analysis indicates that all of the eleven electrophoretic forms consist of a single subunit with a molecular weight of 15.5kDa. These isozymes appear to be produced post-translationally.

Purification and characterization of a lysozome from wheat germ

Several proteins of wheat germ were able to lyse Micrococcus luteus cells. One lysozyme, named W1A, was purified by ammonium sulfate fractionation, ion-exchange chromatography, gel filtration and preparative polyacrylamide gel electrophoresis (PAGE) under native conditions. The enzyme had a molecular weight of 25 400 as determined by sodium dodecyl sulfate (SDS)-PAGE. The reducing groups released from the lysis of Micrococcus cell walls by W1A lysozyme were N- acetylmuramic acid residues as for hen egg white lysozyme (HEWL). Chitin substrates were hydrolyzed to some extent by this enzyme. With Micrococcus cells as substrate, the pH optimum for W1A lysozyme was 6.0 at an optimal ionic strength of 0.05. Under these conditions, the K m value was 166 mg/l with purified Micrococcus cell walls and the V max value was 0.56 A 540 unit/min at 22°C. W1A lysozyme exhibited the highest lytic activity at 60°C whereas the enzyme was inactive above 90°C. W1A lysozyme was strongly inhibited by poly- l-lysine and glycol chitosan. This is the first report of the presence of multiple electrophoretic forms of plant lysozyme activity as determined by native PAGE.

Site‐directed mutations altering methyl‐accepting residues of a sensory transducer protein

The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methyl-accepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.

Electrophoretic forms of tropomyosin in skeletal muscle

Electrophoretic forms of tropomyosin in skeletal muscle

Isolation and characterization of DNA-dependent RNA polymerase III from Leishmania mexicana and inhibition by purine analogs.

A DNA-dependent RNA polymerase has been isolated and characterized from the parasitic flagellated protozoan Leishmania mexicana. The initial stages of purification utilized high-ionic-strength extraction and protamine sulfate treatment. The enzyme was further purified by differential elution by heparin-Sepharose, DEAE-Sephadex, and carboxymethyl-Sephadex chromatography. Analysis of the chromatographically purified RNA polymerase on nondenaturing gels revealed two electrophoretic forms. The enzyme isolated had characteristics of true DNA-dependent RNA polymerase since it required DNA and all four nucleoside triphosphates for synthesis of RNase-sensitive products. Analysis of ammonium sulfate and metal ion optima, as well as relative activities of the enzyme with Mn2+ versus Mg2+, gave results similar to those reported for other RNA polymerase IIIs in eucaryotes. Formycin A triphosphate was found to be a noncompetitive inhibitor of RNA polymerase III, and cordycepin triphosphate was found to be inhibitory, although the exact mode of inhibition was not determined.

Sugar transport by the bacterial phosphotransferase system. Reconstitution of inducer exclusion in Salmonella typhimurium membrane vesicles.

The accompanying articles (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 262, 16241-16253; Mitchell, W.J., Saffen, D. W., and Roseman, S. (1987) J. Biol. Chem. 262, 16254-16260) show that "inducer exclusion" in intact cells of Escherichia coli is regulated by IIIGlc, a protein encoded by the crr gene of the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The present studies attempt to show a direct effect of IIIGlc on non-PTS transport systems. Inner membrane vesicles prepared from a wild type strain of Salmonella typhimurium (pts+), carrying the E. coli lactose operon on an episome, showed respiration-dependent accumulation of methyl-beta-D-thiogalactopyranoside (TMG) via the lactose permease. In the presence of methyl-alpha-D-glucopyranoside or other PTS sugars, TMG uptake was reduced by an amount which was dependent on the relative concentrations of IIIGlc and lactose permease in the vesicles. The endogenous IIIGlc concentration in these vesicles was in the range 5-10 microM, similar to that found in whole cells. Methyl-alpha-glucoside had no effect on lactose permease activity in vesicles prepared from a deletion mutant strain lacking the soluble PTS proteins Enzyme I, HPr, and IIIGlc. One or more of the pure proteins could be inserted into the mutant vesicles; when one of the two electrophoretically distinguishable forms of the phosphocarrier protein, IIIGlc Slow, was inserted, both the initial rate and steady state level of TMG accumulation were reduced by up to 40%. The second electrophoretic form, IIIGlc Fast, had much less effect. A direct relationship was observed between the intravesicular concentration of IIIGlc Slow and the extent of inhibition of the lactose permease. No inhibition was observed when IIIGlc Slow was added to the outside of the vesicles, indicating that the site of interaction with the lactose permease is accessible only from the inner face of the membrane. In addition to the lactose permease, IIIGlc Slow was found to inhibit both the galactose and the melibiose permeases. Uptake of proline, on the other hand, was unaffected. The results are therefore consistent with an hypothesis that dephosphorylated IIIGlc Slow is an inhibitor of certain non-PTS permeases.

Cellular competition in the development of ocular tissues in allophenic mice

C57 BL 6 mice exhibit impaired lens development as evidenced by competition studies with allophenic (chimeric) mice. Strain-specific differences in electrophoretic forms of glucose phosphate isomerase were exploited to determine the strain composition of various ocular and nonocular tissues in allophenic mice constructed from C57 BL 6 and DBA 2 strains. As expected, there was variation from one animal to the next in overall mosaic composition, as well as variation among most ocular and nonocular tissues within individual animals. In contrast, when individual lenses from the same animals were assayed there was a marked and consistent deficiency of the C57 BL 6 component. Control experiments indicated that these results are not the consequence of peculiarities of marker enzyme expression in the lens. Since lens formation occurs early in embryogenesis any anomaly at this stage could influence the normal induction of later ocular structures. We believe that the impaired lens-forming capacity of C57 BL 6 mice may be the underlying cause of the 5–10% frequency of overt ocular defects in this strain.

Biochemical and immunological properties of different electrophoretic forms of juvenile hormone esterase from Trichoplusia ni (Hübner)

The two major electrophoretic forms (p I 5.5, 5.3) of juvenile hormone esterase were independently isolated from hemolymph of larval Trichoplusia ni. A simple and rapid preparation procedure of poly(ethylene glycol) precipitation, Sephadex gel filtration and chromatofocusing is described. Analytical isoelectric focusing showed only one peak of juvenile hormone esterase activity in the respective purified samples, whereas there were four (two major) such peaks in the hemolymph. The amino acid composition of the two forms was similar. The comparison of peptides obtained after protein fragmentation bycyanogen bromide showed that juvenile hormone esterases A and B were very similar, although definitely not identical, in amino acid sequence. The immunological comparisons of juvenile hormone esterases suggested that the number of polyclonal antibody binding sites on both forms was the same. There were no detected differences between immunoreactive properties of juvenile hormone esterase from the hemolymph of different stages of larval maturation. The influence of the active site of the enzyme on its antigenic properties was studied by immunocompetition. The inactive, heat-denatured juvenile hormone esterase can only partially protect against inhibition of its activity by the antibodies, whereas an organophosphate inhibitor which covalently binds to the catalytic center of the enzyme did not change the immunoreactive properties in comparison to active juvenile hormone esterase from hemolymph. These data show that heat-denatured juvenile hormone esterase has lost at least one or more epitopes, but the catalytic site of the enzyme is distinct from the epitopes.

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.

The isolation of the two electrophoretic forms of cowpea mosaic virus using fast protein liquid chromatography

This report describes the first time entire viral capsids have been purified using fast protein liquid chromatography (FPLC) techniques. The FPLC is used here to separate the two electrophoretic forms of cowpea mosaic virus. The capsid forms are shown to be separated by the Mono-Q column without damaging the capsids.

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis.

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc1 proteins. These results show that evolution at the DBP level can be considered as a continuous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.