Characteristics of glutamate dehydrogenase, a new diagnostic marker for the genus Fusobacterium

Enzymes representative of carbohydrate and nitrogen metabolism were screened for their presence and activity amongst species of the genus Fusobacterium. Glutamate dehydrogenase (GDH) was reliably detected in all 25 strains studied. The pH profile of this enzyme and the DNA base composition of selected strains were also determined. DNA base composition of selected strains ranged between 28-32.9 mol% G + C. GDH was active between pH 7.5-11.5 but two pH profiles of activity, with optima at 9.5 and 10.5, were discernible among species. Apart from Fusobacterium nucleatum, which had a heterogeneous enzyme pattern, the GDH electrophoretic mobility was constant within a species but in a few cases the enzyme bands overlapped. A combination of the pH profile, the GDH electrophoretic pattern and the DNA base composition provided clear separation of the test organisms into discrete groups; however, a larger number of strains must be examined before the full potential of these tests can be evaluated.     

Different Forms of Adrenal Phenylethanolamine N-Methyltransferase: Species-Specific Posttranslational Modification

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.     

Different deoxyribonucleases in human lymphocytes

The distribution pattern of deoxyribonuclease activities in human lymphocytes has been examined by micro-disc-electrophoresis. Four groups of deoxyribonuclease activities, differing in their electrophoretic mobility, in the nature of their optimal substrate and in their optimal incubation conditions, are characterized. There are two alkaline DNase-activities. One corresponds to DNase I (EC 3.1.4.5), the other having pH optimum of about pH 9.0, prefers denatured DNA as substrate and is not dependent on divalent cations. The fractions with an acid pH optimum can be subdivided into two groups, which differ in their activity towards native DNA, towards denatured DNA, in their activity when succinate is present and in their pH optimum.     

Macromolecular and ultrastructural organization of the mitotic chromosome scaffold

Using electron microscopy (EM), we have examined three structural domains of the mitotic chromosome scaffold of mouse erythroleukemia (MEL) Friend cells with different morphologic organization: centromeric, intermediate, and telomeric. The intermediate, most extensive, domain exhibited a specific fibrogranular structure representing tightly packed granular bodies with diameters between 20 and 60 nm. The chromosome scaffold contained three main components: proteins (81%), RNA (12%), and DNA (7%). The residual DNA extracted from the scaffold represented short fragments, 300 bp on average, belonging to the class of tandemly arranged repetitive DNA. In situ hybridization experiments confirmed its typical centromeric location. Scaffold RNA represented three fractions: a major RNA fraction with an electrophoretic mobility corresponding to that of 5S RNA and two minor fractions with electrophoretic mobilities somewhat lower than that of 18S RNA. Scaffold RNA was localized mainly in the centromeric region. We show that the newly synthesized protein component of the chromosome scaffolds migrates slowly to the chromosomes, reaching a maximum specific radioactivity 12 h from the onset of the chase period.     

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.     

Detection of beta-amyloid peptide aggregation using DNA electrophoresis

DNA could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of beta(1-40) and beta(25-30). DNA from different sources such as lambda phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric beta(1-40) and beta(25-35) as well as by the aggregated beta(1-40) and beta(25-35). The association of DNA with the aggregated beta-amyloid peptides could occur at pH 4-9. The inhibitory action of hemin on beta-amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of beta-amyloid aggregation as well as for testing of agents that might modulate beta-amyloid aggregation.     

Ku polypeptides synthesized in vitro assemble into complexes which recognize ends of double-stranded DNA.

The Ku protein is composed of two polypeptide subunits, p70 and p80, and binds DNA ends in vitro. Previous studies suggested that p70 and p80 are physically associated in vivo, although such an association may have been mediated by DNA. We have now utilized full-length Ku polypeptides synthesized in vitro to examine the association of p70, p80, and linear DNA to form a complex. In gel filtration chromatography, p70 migrates as a 70-kDa structure, whereas p80 migrates at 150 kDa. Co-translation of the two cDNAs yields complexes which migrate at 300 kDa and contain equimolar quantities of the p70 and p80 polypeptides, providing direct evidence that p70 and p80 assemble into a complex in the absence of DNA. To demonstrate that this recombinant protein complex binds DNA, we developed a radiolabeled protein electrophoretic mobility shift assay. When radiolabeled proteins synthesized in vitro were incubated with linear DNA and fractionated in a nonreducing, nondenaturing gel, a band representing a complex of p70, p80, and the DNA was seen. Formation of this Ku-DNA complex required free DNA ends, and binding to DNA ends was not observed with individual p70 or p80 subunits. DNA binding was not reconstituted by mixing the individual subunits together. These studies thus demonstrate that it is the complex of p70 and p80, not individual p70 or p80, which possesses the DNA binding properties previously described for native Ku protein. These results provide new information about the assembly, structure, and DNA binding properties of the Ku protein.     

Studies on purified α-actinin : I. Effect of temperature and tropomyosin on the α-actinin/F-actin interaction

α-Actinin, purified by two passages through a DEAE-cellulose column, migrates either as a single band or as a single major band with a fainter trailing band during polyacrylamide gel electrophoresis at pH 8.3. In the presence of sodium dodecyl sulfate, purified α-actinin always migrates as a single electrophoretic zone during polyacrylamide gel electrophoresis. Temperature has large effects on the interaction of α-actinin with F-actin. At 0 °C, α-actinin causes large increases in F-actin viscosity, either in the presence or absence of tropomyosin. Quantitative binding studies show that α-actinin can displace tropomyosin from F-actin at 0 °C and that F-actin will quantitatively bind 45% of its weight of α-actinin either in the presence or absence of tropomyosin. This binding ratio corresponds to one α-actinin molecule to approximately 10 to 11 G-actin subunits and suggests that one molecule of α-actinin binds to each turn of the F-actin helix at 0 °C.     

Disappearance of the negative charge in giant DNA with a folding transition

In the present study we measure the electrophoretic mobility of giant T4 DNA (166 kbp) by electrophoretic light scattering for the elongated and folded compact states at different spermidine (trivalent cation) concentrations in 50 mM sodium maleate buffer (pH 6.0). It is found that the electrophoretic mobility of elongated DNA in the absence of the multivalent cation is seven times greater than that of fully folded compact DNA, where, with the increase of the concentration of spermidine, an abrupt transition is generated after a gradual decrease of the mobility. An analysis of the electrophoretic mobility suggests that the folded compact DNA chains almost completely lose their negative charges, by taking into account the difference of friction mechanism between an elongated and folded compact state. From the single chain observation by use of fluorescence microscopy, it is found that a phase-segregated structure is generated at intermediate concentrations of spermidine. The gradual decrease of the electrophoretic mobility in the transition region is, thus, attributed to the formation of the segregated state, exhibiting partial electroneutralization in the folded part. Disappearance of the negative charges in the completely folded compact DNAs is discussed in relation to the mechanism of transition, in terms of a first-order phase transition.     

Asymmetric structure of a three-arm DNA junction

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different conformations of ribosomal DNA in active and inactive chromatin in Xenopus laevis

The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.     

Electrokinetic behavior of inside-out vesicles from human red cell membranes

The electrokinetic behavior of red cell membrane vesicles of normal (ROV) and inverted (IOV) sidedness has been characterized using the laser Doppler technique of electrophoretic light scattering (ELS). At neutral pH ROV have a (approx. 25%) higher electrophoretic mobility than IOV and the two peaks can be resolved in the ELS spectrum to provide a quantitative estimate of the IOV/ROV ratio which is consistent with the ratio determined by assay of the activity of acetylcholinesterase. The ROV peak coincides with the mobility of fresh red blood cells and of resealed ghosts. Neuraminidase treatment reduces the ROV mobility by a factor of 2.6, while the IOV peak is reduced only slightly (<5%). Treatment with trypsin results in a single narrow ELS peak at about 60% of the mobility of ROV. Treatment of IOV with phospholipase C leaves the electrophoretic mobility unaltered, whereas treatment with phospholipase D increases their mode mobility by 22%. The mobility titration curve of IOV from pH 2 to pH 10 reveals three distinct inflection points which may be assigned to chemical groups on the cytoplasmic surface of the red cell membrane.     

Interaction of very low density lipoproteins (VLDL) with rabbit C-reactive protein

Rabbit CRP is similar to human CRP in structure, kinetics of appearance, and binding reactivities to phosphate esters and cationic polymers. CRP in rabbit acute-phase serum migrates either with gamma or with beta, pre-beta electrophoretic mobility, and distinct gamma- and beta-migrating species can be observed simultaneously in some sera. The present study shows that beta-CRP in serum is converted to gamma mobility during isolation and purification. Normal, acute-phase, or CRP-depleted acute-phase rabbit serum restores the beta mobility of purified gamma-CRP, a conversion that does not occur in the presence of EDTA. Serum CRP fails to adsorb to DEAE-cellulose but does adsorb to CM-cellulose, from which it elutes as gamma-mobility antigen. Chelation by EDTA or flotation and removal of lipoproteins from acute phase rabbit serum produces a gamma-mobility CRP that adsorbs to the anion-exchange resin. Lipid-containing fractions from ion-exchange columns as well as VLDL (but not LDL or HDL) isolated by ultracentrifugation change the mobility of purified CRP from gamma to beta, pre-beta. These changes in mobility are not observed in the presence of EDTA or phosphocholine. In acute-phase rabbit serum with CRP of both beta and gamma mobility, the beta form has a higher m.w. and is lipid-associated, whereas the gamma form is a lower m.w., lipid-poor molecule. These results suggest that in serum the association of CRP with lipoproteins, particularly VLDL, is responsible for its beta, pre-beta electrophoretic mobility. Further studies of the association of CRP with lipoprotein in relation to lipoprotein metabolism may provide insight into the biological role of CRP.     

Replication of DNA minicircles in kinetoplasts isolated from Crithidia fasciculata: structure of nascent minicircles.

We have previously described an isolated kinetoplast system from Crithidia fasciculata capable of ATP-dependent replication of kinetoplast DNA minicircles (L. Birkenmeyer and D.S. Ray, J. Biol. Chem. 261: 2362-2368, 1986). We present here the identification of two new minicircle species observed in short pulse-labeling experiments in this system. The earliest labeled minicircle species (component A) contains both nascent H and L strands and is heterogeneous in sedimentation and electrophoretic migration. Component A has characteristics consistent with a Cairns-type structure in which the L strand is the leading strand and the H strand is the lagging strand. The other new species (component B) has a nascent 2.5-kilobase linear L strand with a single discontinuity that mapped to either of two alternative origins located 180 degrees apart on the minicircle map. Component B could be repaired to a covalently closed form by Escherichia coli polymerase I and T4 ligase but not by T4 polymerase and T4 ligase. Even though component B has a single gap in one strand, it had an electrophoretic mobility on an agarose gel (minus ethidium bromide) similar to that of a supercoiled circle with three supertwists. Treatment of component B with topoisomerase II converted it to a form that comigrated with a nicked open circular form (replicative form II). These results indicate that component B is a knotted topoisomer of a kinetoplast DNA minicircle with a single gap in the L strand.     

Electrophoresis of adsorbed DNA

The electrophoresis mobilities of native calf thymus DNA adsorbed on the charged solid particles were measured by a micro-electrophoretic method as functions of pII, ionic strength, and DNA concentration. The mobility data confirm the adsorption of DNA both on the positively charged alumina and negatively charged resin particles at wide range of pH and ionic strength. The mobility data also indicate significant DNA adsorption by negatively charged glass in the acidic range of pH. The electrophoretic mobilities of DNA adsorbed on different substrate particles under identical conditions do not differ widely, indicating the major role of the adsorbed DNA rather than the covered substrate in controlling the charge behavior of the particle. The mobilities of the adsorbed DNA at salt pH are of a comparable order of magnitude to those for the dissolved DNA in solution. The mobility of the adsorbed heat-denatured and alkali-denatured DNA is lower than that of the native adsorbed DNA under identical conditions of pH and ionic strength.     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.     

Analysis of branched nucleic acid structure using comparative gel electrophoresis

Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.     

Electrophoretic analysis of the estrogen receptor. Relationship of multiple receptor forms to the molybdate-stabilized form

The origin of and relationships among multiple forms of the estrogen receptor from rat uteri were investigated using electrophoretic and conventional hydrodynamic methods of analysis. Evidence is presented that the molybdate-stabilized, multimeric receptor (Stokes radius approximately 70A; S20,w approximately 9.5 S; Mr approximately 290,000) corresponds to an acidic form of the receptor that has relatively high electrophoretic mobility. This discrete form, which appears to represent the untransformed state that does not bind to DNA, was converted to a number of derived forms by exposure to conditions that result in receptor transformation and/or subunit dissociation. In crude cytosol, transformation always generated receptor forms that were excluded from polyacrylamide gels, and it was shown that these are large heterogeneous aggregates. This explains previous failed attempts to analyze the receptor by polyacrylamide gel electrophoresis. Transformation of partially purified, molybdate-stabilized receptor never led to aggregate formation, but resulted instead in the generation of two relatively basic estrogen-binding species of low electrophoretic mobility. These components may represent the free or dissociated estrogen-binding subunits. Together, the results suggest a model for the molybdate-stabilized receptor wherein at least one of its components is an acidic, nonestrogen-binding subunit.     

Free solution mobility of oligomeric DNA

The free solution electrophoretic mobility of a charged oligomer in an ionic solvent that approximately takes into account relaxation field effects, screening of the velocity field, and the hydrodynamic interactions resulting from motions of the charges due to an electric field is described. For double‐stranded DNA, the free solution electrophoretic mobility under ionic strengths determined by the buffer and pH conditions relevant to capillary electrophoresis increases with increasing molecular weight up to few hundred base pairs. © 1999 John Wiley & Sons, Inc. Biopoly 49: 209–214, 1999     

Comparative electrophoretic profiles of esterases, and of glutamate, lactate and malate dehydrogenases, from Aeromonas hydrophila, A. caviae and A. sobria

Esterases, and glutamate, lactate and malate dehydrogenases of 64 Aeromonas hydrophila, A. caviae and A. sobria strains, were analysed by polyacrylamide agarose gel electrophoresis and by thin layer isoelectrofocusing. On the basis of the isoelectric points of malate dehydrogenase from the three species and the mobility of lactate dehydrogenase from A. sobria, 8 species specific zymotypes were defined: three for A. hydrophila strains, three for A. caviae strains and two for A. sobria strains. These zymotypes correlated with previously established DNA hybridization groups. The other electrophoretic data were found to be less useful for distinction between A. hydrophila and A. sobria strains, but supported differentiation into zymotypes for A. caviae strains. The two-dimensional electrophoretic profile established by plotting isoelectric point against electrophoretic mobility of the major esterase illustrated the degree of enzyme polymorphism among the strains of the three species. Variation in electrophoretic patterns within A. hydrophila and A. caviae might provide useful epidemiological markers.