Gramicidin S-synthetase. Electrophoretic characterization of the multienzyme.

1. A method characterizing the fully active gramicidin S-synthetase (EC. 6.3.2.-) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. The molecular weight of 280000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300000 daltons has been determined, correlating either mobilities or retardation coefficients.     

Fractionation of chondroitin sulfate and determination of molecular weight by polyacrylamide gel electrophoresis

Ten discrete fractions of chondroitin 4-sulfate were prepared and refined by polyacrylamide gel electrophoresis. The molecular weights were determined based on the molar ratios of the components to the end group xylose. They have the same free electrophoretic mobility and the logarithms of their molecular weights were linearly related to the relative electrophoretic mobilities in the disc polyacrylamide gel electrophoresis. Using some of the fractions as standards, the polydispersity of the chondroitin sulfate was proven to be mainly due to a continuum of varying chain length.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 as a novel approach to actinomycete taxonomy: application to the genera Actinomadura and Microtetraspora.

Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura, and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30 preparations from several strains proved that we were indeed dealing with the specified protein homologous to ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.     

Electrophoretic heterogeneity of ribosomal protein AT-L30 among actinomycete genera.

The ribosomal proteins from 17 type strains of species belonging to various actinomycete genera were compared by two-dimensional polyacrylamide gel electrophoresis. I detected a striking variability among certain ribosomal proteins (designated AT-L30 proteins) with respect to electrophoretic mobility in the first dimension. In contrast, such variability was not observed among ribosomal L30 proteins from other bacteria, such as Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Although actinomycete AT-L30 proteins from different taxa exhibited considerable heterogeneity in electrophoretic mobility, within each genus the proteins had a specific mobility characteristic. On the basis of this observation, the ribosomal AT-L30 proteins from 11 type strains of species belonging to the mycolic acid-containing genera Nocardia, Rhodococcus, Gordona, and Tsukamurella were analyzed. The relative electrophoretic mobilities of AT-L30 protein preparations from these strains, as determined by two-dimensional gel electrophoresis, revealed that the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella can be sharply separated from each other. My results are consistent with the previously discussed view that each of these genera merits separate genus status.     

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

Quantitative comparative analysis of complex two-dimensional electropherograms

A protocol is described to detect and assess differences between complex electrophoretic patterns. A semiautomated method is used to collect accurate absolute mobility data from many two-dimensional electropherograms and a computer algorithm has been developed which normalizes and averages these data. The program generates refined numerical maps consisting of the mean electrophoretic mobilities and corresponding confidence limits for each component protein represented in the original two-dimensional electrophoretic pattern. Tests of statistical significance of apparent differences between averaged numerical maps are carried out to evaluate electrophoretic polymorphisms between the ribosomal proteins of two different plant species. Furthermore, using a nonlinear function relating log molecular weight to mobility, precise molecular weight estimates are obtained from measurements of electrophoretic mobilities of proteins in the presence of sodium dodecyl sulfate. Several examples are presented which demonstrate application of these semiautomated analyses to quantitative comparison and interpretation of two dimensional gel electropherograms.     

The relationship of molecular weight to electrophoretic mobility of fluorescamine-labeled proteins in polyacrylamide gels

Proteins of known molecular weights were labeled with fluorescamine and then subjected to electrophoresis through polyacrylamide gels. The electrophoretic mobilities of the fluorescamine-labeled proteins were dependent upon their respective molecular weights over a range of 17,000 to 70,000 daltons. The correlation of electrophoretic mobility of fluorescamine-labeled protein to molecular weight was similar to results obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The speed with which data can be obtained with the described procedure is a definite advantage over currently employed procedures. These findings encourage the use of fluorescamine for rapid, sensitive determinations of molecular weights of proteins in polyacrylamide gels.     

Adenosine dimethylation of 16S ribosomal RNA: effect of the methylgroups on local conformational stability as deduced from electrophoretic mobility of RNA fragments in denaturing polyacrylamide gels.

The electrophoretic mobility of RNA fragments derived from the 3'-end of 16S rRNA on slabs of polyacrylamide gel in the presence of urea is strongly influenced by dimethylation of the N6-aminogroup of two adjacent adenosines. This is not due to the presence of the methylgroups per se, but must be ascribed to an effect of methylation on long range intramolecular interactions at these denaturing conditions. When it is assumed that the electrophoretic mobilities of the RNA fragments in the polyacrylamide matrix are determined by the conformational state(s) of the fragments, dimethylation of the adenosines leads in the smaller fragments to a less compact average conformation and in the larger fragments to a more compact average conformation. An effort is made to comprehend the effects of adenosine dimethylation in terms of secondary structure based on nucleotide sequence.     

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.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Microspectrophotometric and enzymatic analyses of fish plasma proteins electrophoretically separated in thin polyacrylamide gels

A system for horizontal, discontinuous electrophoresis in thin sheets of polyacrylamide gel was developed to permit rapid and direct comparison of multiple samples of fish plasmas for population studies. The resulting electropherograms are suitable either for screening for enzyme polymorphisms or for densitometric scanning after gels are stained for total proteins. Each gel sheet (110×90×0.8 mm) can be used for simultaneous separation of 10–15 different samples. Since total voltage and the running time required for successful resolution of fine protein bands are greatly reduced in this system, minimal heating occurs within the gel matrix during electrophoresis. Up to 120 different blood samples (8 gel sheets) can be processed in about 8 h. Gel sheets are routinely stained overnight in Coomassie Brilliant Blue (0.1%, v/v), rinsed several times and stored overnight in 7% acetic acid to complete destaining. Particular gel sectors are then transferred to distilled water and mounted on glass microslides for photography or for densitometric evaluation with a Leitz microspectrophotometer. These procedures have been used to identify characteristic differences in the electrophoretic mobilities of plasma enzymes and albumin fractions from several populations of poeciliid fishes. Observed differences in albumin mobilities (albumin phenotypes) were verified by mixing isoaliquots of test plasmas with plasma samples containing albumins of known mobility. Resultant patterns for albumin bands for such mixed plasmas were indistinguishable from those obtained with plasma samples from the F₁ hybrid progeny of parents possessing albumins of characteristically different electrophoretic mobilities. Procedural details for gel casting, electrophoresis and sample evaluation are described.     

Electroendosmosis correction for electrophoretic mobility determined in cells

Electroendosmosis is a complicating factor in gel electrophoresis. Determination of electroendosmotic mobility by the use of vitamin B₁₂ as a marker in agar and agarose gels at different concentrations revealed that electroendosmosis was not reduced to zero by extrapolation of observed mobility values to zero gel concentration. It is shown that a Ferguson plot of the observed values of electrophoretic mobilities yields the correct values for K_R; however the extrapolated values of electrophoretic mobility must still be corrected for electroendosmosis to obtain the true electrophoretic mobilities.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Reduction of Disulphide Bonds at Acid pH and Quantitative Estimation of Prolamins from Rye, Wheat and Barley following Separation by Electrophoresis

Storage proteins from three cereal species were reduced at acidpH in the presence or absence of urea. Reactions were usuallycarried out at 100°C and pH 3.2, using dithiothreitol asthe reductant. Changes in electrophoretic mobility of prolaminsin polyacrylamide gels were taken as evidence of protein reduction.One-dimensional polyacrylamide gel electrophoresis, densitometricanalyses and computerized evaluation of data were used to quantifyreactions. Parameters tested include the influence of time,heat, reductant concentration and the presence or absence ofurea. More than 45% of secalin, the alcohol-soluble proteinfrom rye, readily underwent reductive cleavage to yield bandsof substantially different mobilities. Results suggest thatat least 35% of the alcohol-soluble rye components contain intermoleculardisulphide bonds and that at least 12% consist of intramoleculardisulphides; other components appear to be already fully reducedor devoid of disulphide linkages. Analogous measurements forgliadins and hordeins showed that at least 10% of these prolaminsunderwent similar reductions; mobilities of the products suggestthat intramolecular linkages are involved. The method employedprovides a convenient measure of both the qualitative and quantitativechanges associated with the reduction of protein disulphidebonds and the results are important to an understanding of themolecular reactions responsible for the properties of glutenproteins. Key words: Rye proteins, Wheat proteins, Barley proteins, Reduction, Quantitative PAGE, Densitometry     

Influence of a cationic detergent on electrophoresis in polyacrylamide gel.

Proteins were subjected to polyacrylamide gel electrophoresis at pH ～ 4 in the presence of the cationic detergent HPCl. The relative electrophoretic mobilities of the proteins were correlated to their molecular weight.     

Further Characterization of the Reduced Nicotinamide Adenine Dinucleotide Phosphate:Nitrate Oxidoreductase in Aspergillus nidulans

The reduced nicotinamide adenine dinucleotide phosphate (NADPH):nitrate oxidoreductase (EC 1.6.6.2) from Aspergillus nidulans wild-type bi-1 was purified by means of salt fractionation, gel filtration, affinity chromatography, and polyacrylamide gel electrophoresis. Enzyme which was adsorbed on Cibacron blue agarose could be eluted with 2 mM NADPH or 2 mM oxidized NADP (NADP(+)), the former being about three times more effective than the latter. About half the total NADPH:nitrate reductase activity adsorbed on agarose required elution with 1 M NaCl. This salt-elutable form remained active with NADPH and was not converted to the NADPH-elutable form after readsorption on Cibacron blue agarose. The NADPH-eluted enzyme exhibited a markedly different electrophoretic mobility than the enzyme eluted with NADP(+) or NaCl. After electrophoresis on polyacrylamide gels, the NADPH-eluted NADPH:nitrate reductase was separated into four proteins, two of which contained nonheme iron and exhibited reduced methyl viologen-nitrate reductase activity. None of these proteins, singly or in combination, reduced nitrate with NADPH as substrate. Difference spectra analyses and specific heme iron stains revealed the presence of cytochrome b(557) in the largest of the proteins. The molecular weights of the four proteins, which were determined from the relationship of their mobilities on varied concentrations of acrylamide gel, were 360,000, 300,000, 240,000, and 118,000. The subunit molecular weights of these, which are determined via sodium dodecyl sulfate slab gel electrophoresis, were 49,000, 50,000, and 75,000. The key role of NADPH in maintenance of the active form of the heteromultimer is further substantiated.     

Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions

Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.     

The histone H5 variant in Xenopus laevis

The presumptive histone H5 of Xenopus laevis has been characterized by SDS and acid-urea-Triton polyacrylamide gel electrophoresis and compared with chicken histone H5. Chicken H5 has a lower electrophoretic mobility compared to that of Xenopus H5 in both gel systems. It is shown, using a polyclonal antiserum against chicken H5, that the Xenopus histone H5 is immunologically related to chicken histone H5. Monoclonal antibodies have been prepared to the Xenopus histone types H5 and H1A, that do not cross-react, as determined by their reactivity in an enzyme linked immunosorbent assay and by their ability to react with either H1A or H5 in an immunochemical test on total erythrocyte histones that are transferred to nitrocellulose after fractionation by SDS- or acid-urea polyacrylamide gel electrophoresis. As all nuclei of erythrocytes from adult Xenopus laevis can be shown to contain histone H1A and H5, these monoclonal antibodies can be used to further delineate the role of H5 in tissue differentiation.     

Analysis of polypeptide molecular weights by electrophoresis in urea

Ten proteins of differing disulfide contents and isoionic points were subjected to disc gel electrophoresis in the presence of 8 urea-0.9 acetic acid to evaluate the use of this technique in determining polypeptide molecular weights. Comparison of the electrophoretic mobilities before and after reduction of the proteins' disulfide bonds demonstrated that only after all disulfide bonds were broken, could their molecular weights be estimated with any degree of accuracy. The expression of the electrophoretic mobilities as a function of the proteins' effective hydrodynamic sizes, thereby taking into account the extent of constraint by disulfide bonds, allowed a comparison of disulfide cross-linked and linear forms of the protein polypeptides. The extent to which intrinsic charge affects a protein's electrophoretic mobility was estimated by comparing alpha-lactalbumin and lysozyme, two proteins of identical size but vastly different isoionic points. They exhibited a 20% difference in mobilities. An apparent slow reduction of disulfide bonds was observed to occur when proteins were exposed to reducing agent at low pH in 8 urea.     

Ribosomal protein differences between animal cells

Ribosomal proteins of human (HeLa), Syrian hamster, Chinese hamster, chick (embryo) and rat (Novikoff hepatoma) cells have been examined by two-dimensional polyacrylamide gel electrophoresis. The results show that although there are many similarities between the electrophoretic patterns, species-specific marker proteins can be identified for Syrian hamster, chick, rat and possibly HeLa cells, which could be used in genetic analysis. No specific protein marker has been identified for Chinese hamster. The similarity in electrophoretic mobility of the hamster, chick and rat marker proteins suggests an overall structural relationship between them.