Isolation and preliminary characterization of electrophoretic variants of copper,zinc superoxide dismutase

Summary Three electrophoretic variants of superoxide dismutase can be detected in bovine erythrocytes by gel electrophoresis and electrofocusing. The two major forms, having isoelectric points at pH 5.2 and 4.9, were isolated by preparative focusing or chromatography. No differences were found in molecular weight, metal content, antigenicity, electron spin resonance spectrum, visible and ultraviolet optical spectra. In contrast, holo- and apo-superoxide dismutase, which have an electrophoretic mobility similar to that of the two major forms, showed unresolved isoelectric points but significantly different antigenicity. This result suggests that their different electrophoretic mobility is mainly conformation-related. The variant with pl 5.2, corresponding to the protein purified by ordinary procedures, was found to be inactivated by heat treatment faster than the other form. The latter one, on the other hand, gave rise to a multiple pattern of electrophoretic bands after incubation at 75 °C.It is suggested that superoxide dismutase multiplicity in erythrocytes is not genetically determined, but may be related to segregation of subunits, made non-identically by post translational asymmetrical modification.     

Relation between reduced electrophoretic mobility of human lymphocytes and the inhibition of spreading of human monocytes caused by the same anti-human lymphocyte globulin

Slowing of electrophoretic mobility of human peripheral blood lymphocytes after adding anti-human lymphocyte globulin (AHLG) in vitro correlated with indices of the monocyte spreading inhibition obtained by the same AHLG. Consequently, electrophoretic results may be associated with the prolongation of skin allograft survival in primates - the best and the only practical in vivo test for determination of the immunosuppressive potency of AHLG. If so, the simple electrophoretic test could be used in combination with other in vitro tests, preferably for monitoring of the AHLG production.     

Cellular Electrophoretic Mobility and the Mitotic Cycle

The electrophoretic mobility of RPMI No. 41 cells grown in suspension, parasynchronized by double thymidine blocking and cold shock, is reported. No. 41 cells have a higher electrophoretic mobility during the mitotic peak phase than at other times in the mitotic cycle. Treatment of parasynchronous cells by neuraminidase reduces the mobility to the same value irrespective of the stage of the cells in the mitotic cycle. The higher electrophoretic mobility of cells in mitotic peak phase is probably due to a higher surface charge density at this time, possibly caused by a higher concentration of ionized neuraminic acid carboxyl groups at the hydrodynamic shear layer. The mobility of nonsynchronous rapidly and slowly growing cells differs; neuraminidase reduces their mobility by proportionately similar amounts. The results suggest that the differences in mobility between rapidly and slowly growing cells cannot be accounted for exclusively by differences in the amount of neuraminic acid groups at the shear layer.     

Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles

Brain creatine kinase is principally of soluble cytoplasmic origin (anodal electrophoretic mobility). However, synaptosomal membranes and synaptic vesicles are enriched in an isoenzyme electrophoretically similar to muscle type creatine kinase (cathodal electrophoretic mobility), but which can be distinguished from muscle type by other means.     

Effect of ribonuclease and neuraminidase on the electrophoretic mobility of tissue culture cells in parasynchronous growth

Cultured mammalian cells (RPMI no. 41) in parasynchronous growth were treated, at different stages of the mitotic cycle, with neuraminidase and ribonuclease, separately and sequentially, and their electrophoretic mobilities determined. Changes in the electrophoretic mobility of these cells are probably mainly due to variations in the density of negatively charged groups susceptible to neuraminidase, although variations in groups susceptible to ribonuclease may occur. It is suggested that the observed variations in electrophoretic mobility of different cells may be due to differences in the relative lengths of different life-cycle phases. Where G₂ phase is relatively long or G₁ relatively short the cell populations will hve higher mean electrophoretic mobilities.     

Analysis of electrophoretic mobility histograms of mouse splenocytes and thymocytes during tumor growth and after combined chemotherapy and immunotherapy

Changes in electrophoretic mobility histograms of splenocytes and thymocytes were studied in plasmacytoma X5563-bearing mice as an indicator of response to treatment with mitomycin C (MMC) alone or combined with the immunomodulator Krestin (PSK). Tumor growth was inhibited by 80-90% in the MMC-treated and was further inhibited in the MMC and PSK-treated group. Electrophoretic mobility histograms of splenocytes were used to determine the fraction of cells having intermediate mobility between high mobility (T cells) and low mobility (B cells). This fraction of intermediate-mobility cells increased in tumor-bearing mice, but a normal electrophoretic mobility pattern was obtained following successful antitumor treatment. In the electrophoretic mobility histogram of thymocytes, on the other hand, the low-mobility cells (cortical thymocytes) decreased in number during tumor growth and were further reduced in the MMC-treated group. This reduction was less in the MMC and PSK-treated group. These results suggest that combined therapy with MMC and PSK prevents damage of the host defence mechanism and allows more efficient antitumor treatment. Analysis of electrophoretic mobility histograms of splenocytes and thymocytes using a fully automated cell electrophoretic instrument makes possible the rapid evaluation of the immunological effects of drug therapy of tumor-bearing mice.     

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.     

The stepwise mutation model: an experimental evaluation utilizing hemoglobin variants

The stepwise mutation model of Ohta and Kimura (1973) was proposed to explain patterns of genetic variability revealed by means of electrophoresis. The assumption that electrophoretic mobility was principally determined by unit changes in net molecular charge has been criticized by Johnson (1974, 1977). This assumption has been tested directly using hemoglobin. Twenty-seven human hemoglobin variants with known amino acid substitutions, and 26 nonhuman hemoglobins with known sequences were studied by starch gel electrophoresis. Of these hemoglobins, 60 to 70% had electrophoretic mobilities that could be predicted solely on the basis of net charge calculated from the amino acid composition alone, ignoring tertiary structure. Only four hemoglobins showed a mobility that was clearly different from an expected mobility calculated using only the net charge of the molecule. For the remaining 30% of hemoglobins studied, mobility was determined by a combination of net charge and other unidentified components, probably reflecting changes in ionization of some amino acid residues as a result of small alterations in tertiary structure due to the amino acid substitution in the variant. For the nonhuman hemoglobins, the deviation of a sample from its expected mobility increased with increasing amino acid divergence from human hemoglobin A.-It is concluded that the net electrostatic charge of a molecule is the principal determinant of electrophoretic mobility under the conditions studied. However, because of the significant deviation from strict stepwise mobility detected for 30 to 40% of the variants studied, it is further concluded that the infinite-allele model of Kimura and Crow (1964) or a "mixed model" such as that proposed by Li (1976) may be more appropriate than the stepwise mutation model for the analysis of much of the available electrophoretic data from natural populations.     

An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5( 1.00) allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model.     

Label-free determination of the number of biomolecules attached to cells by measurement of the cell's electrophoretic mobility in a microchannel

We developed a label-free method for a determination of the number of biomolecules attached to individual cells by measuring the electrophoretic mobility of the cells in a microchannel. The surface of a biological cell, which is dispersed in aqueous solution, is normally electrically charged and the charge quantity at the cell's surface is slightly changed once antibody molecules are attached to the cell, based on which we detect the attachment of antibody molecules to the surface of individual red blood cells by electrophoretic mobility measurement. We also analyzed the number of antibody molecules attached to the cell's surface using a flow cytometer. We found that there is a clear correlation between the number of antibody molecules attached to the individual cells and the electrophoretic mobility of the cells. The present technique may well be utilized not only in the field of cell biology but also in the medical and pharmaceutical industries.     

Electrophoretic cell separation by means of microspheres.

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distributions, may be significantly enhanced by immunospecific labeling of target populations using microspheres.     

Immunochemical study of subtilisins obtained from Bacillus subtilis A-50 and some of its mutants]

Physico-chemical parameters of subtilisins from the original Bacillus subtilis A-50 strain (proteolytic activity, electrophoretic mobility, molecular weight, reactions with specific inhibitors) were similar to those mentioned in the literature for the enzymes of other strains. Immunological experiment has shown, that Bacillus subtilis A-50 subtilisins with various electrophoretic mobility do not differ in their antigenic properties. Enzymes with high electrophoretic mobility from mutant strains were similar to I--III subtilisin fractions from Bac. subtilis A-50 in the antigenic characteristics. However, the antigenic heterogeneity was observed in I, II and III enzyme fractions of some mutant strains. Subtilisins studied appear to form the isoenzyme system.     

Electrophoretic cell separation by means of microspheres

The electrophoretic mobility of fixed human erythrocytes immunologically labeled with poly(vinylpyridine) or poly(glutaraldehyde) microspheres was reduced by approximately 40%. This observation was utilized in preparative scale electrophoretic separations of fixed human and turkey erythrocytes, the mobilities of which under normal physiological conditions do not differ sufficiently to allow their separation by continuous flow electrophoresis. We suggest that resolution in the electrophoretic separation of cell subpopulations, currently limited by finite and often overlapping mobility distribution, may be significantly enhanced by immunospecific labeling of target populations using microspheres.     

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.     

Esterase-16 (ES-16) of the rat (Rattus norvegicus): Identification and genetic characterization

Esterase-16, an esterase present in lung and other tissues of the laboratory rat, has been characterized by its biochemical properties (electrophoretic mobility, substrate pattern, sensitivity to inhibitors) and genetic variation in 107 inbred strains and substrains including 14 RI strains. It was classified as a carboxylesterase (EC 3.1.1.1). The phenotype ES-16A (BN/Han and 63 other strains) was defined as a narrow electrophoretic band migrating between ES-1A and ES-13A, ES-16B (LEW/Han and 42 other strains) exhibited the same electrophoretic mobility as ES-16A but was distinguished by its extremely weak activity. Segregation of ES-16 in RI strains and backcrosses indicated linkage to linkage group V (LGV). The Es-16 locus was tentatively placed into esterase cluster 2 and homology with Es-7 of the house mouse is proposed.     

Alterations of the electrophoretic mobility distribution of rat mast cells after immunologic activation.

Changes in the electrophoretic mobility distributions of rat serosal mast cells after immunologic activation have been measured using the laser Doppler technique of electrophoretic light scattering. Rat serosal mast cells of 98% purity isolated by isopycnic and velocity gradient sedimentation had a highly negative electrophoretic mobility which was unaffected by incubation with normal rabbit serum or, at 4 degrees C or in the absence of Ca+2, with rabbit anti-rat E(ab')2 antiserum. Immunologic activation of the cells with this antiserum in the presence of Ca+2 at 37 degrees C resulted in a dose- and time-dependent increase in the electrophoretic mobility. Thus at a 1:25 dilution of anti-F(ab')2 the mean and mode electrophoretic mobilities of the mast cell population increased 25 and 21%, respectively. The width of the electrophoretic mobility distribution also increased with activation, indicating a heterogeneous response of the mast cells in the population. The increase in electrophoretic mobility after immunologic activation is not diminished by treatment of the cells with 1 M NaCl to solubilize adsorbed mast cell granule or heparin.     

A new model for very low density lipoprotein metabolism—Nomenclature for very low density lipoprotein derivatives

A new model for the catabolism of very low density lipoprotein will be proposed following a brief discussion of the relevant information about its metabolism. This model is based on the assumption that each very low density lipoprotein derivative has a distinct structural feature which determines its biological fate. Since this unique determinant may reside in only a small portion of the particles, such as apoprotein, the different very low density lipoprotein derivatives may possess similar or even identical electrophoretic mobility or hydrated density. For this reason, we propose that very low density lipoprotein derivatives be defined according to their putative position in the metabolic pathways rather than according to their hydrated density or their electrophoretic behavior. Thus it is recommended that biochemical separation methods based on principles other than electrophoretic mobility or hydrated density be used to isolate, purify and define very low density lipoprotein derivatives. The position that a lipoprotein particle occupies in the catabolic pathways should be determined by its ability to interact with liver cells or its ability to become converted to low density lipoprotein. This new nomenclature would eliminate unnecessary confusion and stimulate more research toward elucidating the unique structural feature of each very low density lipoprotein derivative.     

A study of baboon hemoglobin.

Electrophoretic analysis of hemoglobin types of 409 baboons of various species, mostly from Senegal, corresponds with the findings of other authors. Baboon hemoglobin is homogeneous as a whole, but differs electrophoretically from that of other monkey species. However, a difference in the electrophoretic mobility of the nonhemoglobin fraction of Papio anubis and Papio cynocephalus suggests a possibly different amino-acid sequence. This information may be useful for the classification in doubtful cases.     

Sialoglycoproteins and sialoglycolipids contribute to the negative surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi have a net negative surface charge, as determined by direct measurement of the mean cellular electrophoretic mobility. Treatment of the parasites with neuraminidase reduces by 17 and 52% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms, respectively. Neuraminidase-treated cells recover their normal electrophoretic mobility if incubated for 2 h in the presence of fresh culture medium. The recovering process of epimastigotes is almost totally blocked by addition of inhibitors of either protein synthesis (puromycin) or N-glycosidically linked glycoprotein synthesis (tunicamycin). The recovering process of trypomastigotes is not totally inhibited by either puromycin or tunicamycin. Treatment of T. cruzi with trypsin reduces by 11 and 40% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms. Trypsin-treated cells recover their normal electrophoretic mobility if incubated for 4 h in fresh culture medium. The recovering process of trypomastigotes is partially inhibited by puromycin. The results obtained indicate that sialoglycoproteins and sialoglycolipids exist on the surface of T. cruzi, the latter being predominant on the surface of trypomastigotes.