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.     

Electrophoretic Heterogeneity of Pigmented Hamster Melanoma Cells

Pigmented hamster melanoma tumors growing in situ contain two subpopulations of melanoma cells that have different electrophoretic mobilities (EPM). A mild neuraminidase treatment, which removes sialic acid residues from the cell surface glycoproteins, reduces the EPM of both groups of melanoma cells yielding an electrophoretically uniform population. This shows that the differences in the EPM between the subpopulations of pigmented melanoma cells stem from the different content of sialic acid residues on the cell surface. The relationship between the different EPM melanoma cell subpopulations was, therefore, examined during tumor growth, development, and formation of metastases. The relative content of cells having high electrophoretic mobility, the “fast moving” cells, increases as the tumors grow larger. However, tumors of the same diameter contain nearly the same fraction of “fast moving” cells despite their age. The proportion of the “fast moving” cells is significantly higher in the central part than in the outermost layer of pigmented melanoma tumors. These data suggest that the development of “fast moving” cells is promoted by some size-dependent changes in the intratumor environment. In vivo selection of melanoma cells for their ability to colonize lungs renders tumors that reveal elevated metastatic potential and contain a significantly higher fraction of cells possessing high electrophoretic mobility than the parent tumor. Moreover, the metastatic nodules contain a remarkably elevated fraction of the “fast moving” cells. The reported correlation between the “fast moving” cell fraction and the metastatic potential suggests that the relative content of cells having high electrophoretic mobility may determine the metastaticity of pigmented hamster melanoma.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Electrophoretic mobility distributions of normal human T and B lymphocytes and of peripheral blood lymphoblasts in acute lymphocytic leukemia: effects of neuraminidase and of solvent ionic strength.

Human T and B lymphocytes were found to be distinguishable on the basis of electrophoretic mobility, with the T cells having the higher mobility, in agreement with previous reports. The effects of the enzyme neuraminidase on the electrophoretic mobilities of T and B lymphocytes were determined. T lymphocytes showed a greater decrease in electrophoretic mobility after neuraminidase treatment; the relative mobilities of T and B cells were reversed by neuraminidase treatment, and the electrophoretic distinguishability was enhanced. The electrophoretic mobility distributions of lymphoblasts from patients with acute lymphocytic leukemia were found to differ from those of normal cells in their response to neuraminidase treatment and to changes in solution ionic strength. These results imply that the surface structure of the leukemic cells differs from that of either T or B lymphocytes from normal donors.     

Storage stability of 14 plasma proteins in ACD-AG banked blood

In summarizing the results it can be stated that, when stored, the proteins haemopexin, alpha 1-antichymotrypsin, alpha 1-antitrypsin, which are rich of neuraminic acid, and IgA are predominantly characterized by a diminution of electrophoretic mobility and thus of concentration in electro-immunodiffusion, with the neuraminic acid content decreasing at the same time. These changes are probably caused by bacteria and/or virus neuraminidase. The decrease of pre-albumin concentration to be observed in radial-immunodiffusion and electro-immunodiffusion is caused by other mechanisms because pre-albumin contains no neuraminic acid. In accordance with these findings the most important changes of single proteins which can be measured by our equipment concern the percentage of non-proteins, whereas the antigen binding places will remain relatively stable during storage (ep. findings of radial immunodiffusion).     

Robonucelic acid within the cellular peripheral zone and the binding of calcium to ionogenic sites

Ribonuclease was shown to reduce the electrophoretic mobility of a line of cultured mammalian cells (RPMI no. 41), and Ehrlich ascites tumour cells. No reduction was detected in the case of human, mouse or embryonic chick erythrocytes. These data, taken with the various controls, support the hypothesis that RNA is a structural component of the peripheries of two types of cells, but not of erythrocytes from three species. Calcium-binding was studied in RPMI no. 41 cells, Ehrlich ascites tumour cells, and human and mouse eryhrocytes, by measurement of reduction in cellular electrophoretic mobility in suspending solutions containing various concentrations of calcium chloride. The effect of treating cells with neuraminidase and/or ribonuclease on calcium-binding was also studied. The results suggest that less calcium binds to the carboxyl groups of peripheral sialic acids than to the phosphates of peripheral, structural RNA. However, calcium apparently binds most avidly to as yet unidentified anionic sites.     

Phosphorylation of the lysine-rich histones throughout the cell cycle.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

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.     

The glycoprotein nature of A1 adenosine receptors

A1 adenosine receptors from different tissues and species were photoaffinity labelled and then the carbohydrate content was examined by both enzymatic and chemical treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labelled membrane receptors shows that neuraminidase treatment alters the electrophoretic mobility of the receptor band indicating the presence of terminal neuraminic acids. Neuraminidase digestion does not influence the binding characteristics of the receptor. The totally deglycosylated receptor protein obtained by chemical treatment has an apparent molecular weight of 32,000.     

ELECTROPHORETIC MOBILITY OF SEA-URCHIN EGGS DURING THE DIVISION CYCLE

The electrokinetic potential of fertilized sea-urchin eggs, without the fertilization membrane and hyaline layer, was investigated by measuring the electrophoretic mobility of the eggs from fertilization to the second cleavage. A cyclic change in mobility was found to accompany the division cycle: the peak of the change was observed about 15 min before the appearance of both the first and second cleavage furrows.
A smaller peak was observed at 20–30 min after fertilization, but such a peak was not repeated between the first and the second cleavage.
Fertilized eggs with the fertilization membrane intact did not show a significant change in electrophoretic mobility throughout the division cycle.     

Phosphorylation-mediated changes in the electrophoretic mobility of CD5 molecules.

This work shows that tumor promoter agents (TPA) induce the post-translational modification of the human lymphocyte surface CD5 antigen (Tp67) in several cellular types. Treatment of [32P]orthophosphate- and [35S]cysteine-labeled normal and lymphoblastoid T and B cells with active tumor promoters induced the rapid, transitory and dose-dependent appearance of hyperphosphorylated CD5 forms with higher apparent molecular masses. These changes in the electrophoretic mobility of CD5 molecules were independent of RNA and protein synthesis, as well as of differences in neuraminic acid content. The inhibition of the TPA-mediated changes by protein kinase C inhibitors (staurosporine and 1-(5-isoquinolylsulfonyl)-2-methylpiperazine) indicated its protein-kinase-C-mediated nature. Phosphatase digestion of CD5 immunoprecipitates reverted the TPA-mediated mobility changes showing its dependence on phosphorylation. Neuraminidase digestion of intact cells revealed that the target of the TPA effects are surface-expressed CD5 molecules. In conclusion, we suggest that the heterogeneity in the electrophoretic mobility induced by TPA could reflect some structural and/or functional differences within CD5 molecules.     

Stability and covalent modification of P-glycoprotein in multidrug-resistant KB cells

An antipeptide antibody (P7) to P-glycoprotein has been produced by immunizing rabbits with a synthetic peptide. Antibody P7 is directed against the amino-terminal region of P170 (residues 28-35). The antibody immunoprecipitates a 170-kDa P-glycoprotein from extracts of drug-resistant KB-V1 cells that is not present in the drug-sensitive cell line KB-3-1. Antibody P7 was used to quantitate the amount of P-glycoprotein present in drug-resistant KB lines at various levels of resistance and to demonstrate the presence of P-glycoprotein in NIH 3T3 cells transfected with a cloned MDR1 cDNA or human genomic DNA encoding MDR1. Pulse-chase labeling experiments demonstrated that P-glycoprotein is synthesized as a 140-kDa precursor which is slowly converted over 2-4 h to a 170-kDa glycoprotein. Tunicamycin treatment blocks the conversion of the precursor to the mature form, and removal of N-linked oligosaccharides with Endo F reduces the relative molecular weight of P-glycoprotein to 140K. The mobility of mature P-glycoprotein is unaffected by treatment with neuraminidase and Endo H. These data indicate that P-glycoprotein is N-glycosylated and contains little or no neuraminic acid. P-Glycoprotein is also phosphorylated, and the extent of phosphate incorporated is proportional to the amount of protein present in drug-resistant cells.     

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae.

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minimum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion and mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle.     

Partial characterization of intra- and extracellular forms of the glycoprotein hemopexin in liver cell culture and cell-free translation

Primary cultures of rat hepatocytes produce extracellular and intracellular species of hemopexin. We examined the presence of high-mannose oligosaccharides and neuraminic acid residues in these species by comparing their electrophoretic mobility on SDS-PAGE before and after digestion by endoglycosidase H and neuraminidase. The predominant intracellular form was not susceptible to digestion by neuraminidase but was sensitive to endoglycosidase treatment, which digested it to a species with a molecular weight comparable to that of the sole hemopexin species produced by tunicamycin-treated hepatocytes and that produced by in vitro translation. By contrast, both the minor intracellular and the extracellular species of hemopexin were neuraminidase-, yet not endoglycosidase H-sensitive, and may be identical. It can be concluded that the intracellular precursor contains high-mannose type oligosaccharides which are processed to complex type oligosaccharides shortly before secretion of hemopexin.     

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 cell cycle of low passage and high passage human diploid fibroblasts

Using mitotic selection, synchronous populations of passage 25 and passage 50 WI38 cells were obtained. Thymidine incorporation, mitotic index, and labeling index were determined. No great differences could be seen in the organization of the cell cycle of low passage and high passage cells. Although labeling indices for passage 25 and passage 50 exponential cells were 83% and 37%, respectively, 75% of all mitotically selected cells were found to enter S phase, regardless of passage number. Thus, the loss of late passage diploid cell cultures does not occur from alteration of the cell cycle in the majority of rapidly proliferating cells.     

Effect of neuraminidase and proteolytic enzymes on electrophoretic mobility of erythrocytes and their aggregation induced by La(3+)

The effect of neuraminidase, trypsin and pronase on the electrophoretic mobility of human erythrocytes and their aggregation induced by La3+ was studied in vitro. The aggregation of intact red cells differs from that of cells treated with neuraminidase and proteolytic enzymes. The above differences are observed in the range of La3+ suspension concentration from 20 to 330 microM. A possible mechanism of aggregation is discussed.     

The effect of neuraminidase on the relative surface charge-associated properties of rat red blood cells of different ages

Approx. 70% of the sialic acid on the rat erythrocyte surface is susceptible to cleavage by neuraminidase (Vibrio cholerae). Neuraminidase treatment results in a reduction in the partition coefficient (K) of the red cells in a charged dextran-poly(ethylene glycol) aqueous phase system and in the electrophoretic mobility of the cells. Countercurrent distribution of rat neuraminidase-treated erythrocytes, containing 59Fe-labeled mature red cells of distinct age, indicates that (a) the electrophoretic mobilities of red cells in different cavities along the extraction train increase with increasing K, as is the case with untreated erythrocytes, and (b) the cell age-related differences in surface charge-associated properties are neither eliminated nor altered by the enzyme action.     

The ionogenic nature of the secretory-granule membrane. Electrokinetic properties of isolated chromaffin granules

1. Chromaffin granules isolated from the bovine adrenal medulla possess an electrophoretic mobility of -1.12mum.s(-1).cm.V(-1), corresponding to a surface zeta potential of -14.4mV and surface charge density of 1.38x10(-6)C.cm(-2). 2. The mobility of chromaffin granules is pH-dependent, indicating an amphoteric surface with an isoelectric point at pH3.0 and acidic groups with a pK(a) of 3.11. 3. Addition of bi- and ter-valent cations decreased the mobility of chromaffin granules in a dose-dependent fashion with a relative potency of La(3+)>Mn(2+)>Ca(2+) >Sr(2+)>Mg(2+)>Ba(2+). 4. Treatment with neuraminidase decreased the mobility of erythrocytes by 84%, whereas chromaffin-granule mobility was decreased by only 14%. This correlates well with the small complement of neuraminic acid present in the granule membrane. 5. The nature, origin and significance of the anionic surface charge of the chromaffin granule is discussed. It is concluded that the net negative charge at the surface of shear derives chiefly from a single type of chemical group, namely -CO(2) (-), contributed by the alpha-carboxyl group of constituent proteins, the phospholipid phosphatidylserine and, to a lesser extent, the sialic acid component of glycoproteins.