Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase)

Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the sialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens$\text{N-}\text{acetylneuraminate}$ glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspension medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca²⁺ in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that a restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.     

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.     

Subcellular fractionation of WI-38 fibroblasts: Compatison between young and old cells

WI-38 fibroblasts cultivated in vitro were homogenized and their subcellular organelles analysed by the techniques of differential centrifugation and isopycnic equilibration in density gradient. In these experiments, the assayed enzymes were known to be specifically associated with subcellular components in other cells types. In most cases, their behaviour and properties corresponded with observations made in earlier studies and we could consider them as being representative of the specific subcellular organelles.Some significant differences were observed between young and old fibroblasts. The specific activity of alkaline phosphodiesterase was lower in the old cells whereas for the other enzymes it was identical or higher, especially for the 5′-nucleotidase; also the particulate fractions obtained by differential centrifugation contained more material. After equilibration in density gradient, the average density of the 5′-nucleotidase, alkaline phosphodiesterase and $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-glucosaminidase}$ was less in the old than in the young cells, whereas that of the galactosyltransferase of Golgi apparatus was greater. For mitochondria, endolasmic reticulum and peroxisomes, the differences observed were small.     

Properties and regional distribution of cerebral CMP-N-acetylneuraminic acid: Glycoprotein sialyltransferase

Glycoprotein sialyltransferase was studied in the rat brain and in the frontal grey cortex and corpus callosum of the calf brain. Activities were measured with endogenous acceptors as well as with desialized α₁-acid glycoprotein as an exogenous acceptor. The enzyme was characterized by means of its pH optimum, K_m values and requirements for detergent and cations. The properties of the rat and calf brain enzymes appeared to be very similar. Substrate specificity studies indicate that more than one glycoprotein sialyltransferase reaction may occur in brain. The regional distribution of the enzyme in the calf brain was rather uniform. From this it was concluded that glycoprotein sialyltransferase, at least for the greater part, is localized in membranes other than those of the synaptic complexes, and occurs in both neurons and glia cells. The regional distribution of the amounts of endogenous glycoprotein acceptor sites, which could be calculated from the sialyltransferase activities, showed a striking correlation with that of the protein-bound sialic acid, but not with the sialyltransferase activity. The role of these endogenous glycoprotein acceptors in cerebral sialoglycoprotein biosynthesis is discussed.     

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Partial $\text{in}\text{vitro}$ sialylation of biantennary and triantennary glycopeptides of α₁-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution ¹H-NMR spectroscopic analysis of the isolated products enabled the assignment of the $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→3)}\text{Man}$ branch as the most preferred substrate site for sialic acid attachment. The $\text{Galβ(1→4)GlcNAcβ(1→2)Man}\text{α(1→6)}\text{Man}$ branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal $\text{N}$-acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.     

A direct evidence for the early membrane desialylation in cobalt-irradiated mouse lymphocytes

Using the sensitive periodate-borotritide radioassay to quantify the membrane sialic acid amount, we show that the $\text{in vitro}$ [⁶⁰Co]-irradiation of mouse splenocytes (10–50 Gy) significantly decrease their membrane sialic acid amount. The results show that the irradiation-induced desialylation is a very early phenomenon since the periodate oxidation is done immediately after the irradiation. A short incubation of cells at 25° C does not increase the extent of the desialylation. This membrane alteration might explain the rapid and drastic decrease in lymphocyte counts in mammals exposed $\text{in vivo}$ to irradiation.     

Rheological characteristics of desialylated erythrocytes in relation to fibrinogen-induced aggregation

The effect of fibrinogen and sialic acid content of erythrocytes on the aggregation of erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. (1) The electrophoretic mobility of erythrocytes was proportional to the sialic acid content of erythrocytes (the surface potential of erythrocytes could be expressed by the sialic acid content). (2) The aggregation of erythrocytes was accelerated by increasing fibrinogen concentration in the medium (due to the increased bridging force among erythrocytes) or by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes). (3) An empirical equation expressing the velocity of aggregate formation (ν, in μm²/min) by the concentration of fibrinogen ($\text{F}$, in g/dl) and the sialic acid content ($\text{S}$, in μmol/ml red blood cells), log $\text{ν = ?0.065 F}{}^{\mathrm{?1.2}}\text{S + 2.2 F}{}^{0.35}$, was deduced. (4) The contribution of the bridging force of fibrinogen to the erythrocyte aggregation was much greater than that of the electrostatic repulsive force produced by sialic acid on the surface of erythrocytes.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

The interaction of ethanol and exogenous arachidonic acid in the formation of leukotrienes and prostaglandin D2 in mastocytoma cells

The present studies were undertaken to examine the hypothesis that ethanol could effect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered $\text{in vivo}$ as well as during $\text{in vitro}$ experiments. The effects of ethanol which encompass these concentration ranges (200–1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20–50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandins D₂ (PGD₂) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 μg/ml), large increases in both PGD₂ and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD₂ formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments sugsests that ethanol affects metabolsim of arachidonic acid at reasonably low doses (200–400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, $\text{in vitro}$ experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500–1000 mg%) which are frequently utilized.     

Sialyltransferase activity in regenerating rat liver

Liver microsomal fractions catalyse the transfer of sialic acid from CMP-N-acetyl-neuraminic acid to various exogenous acceptors such as desialylated fetuin, desialylated human Tamm–Horsfall glycoprotein and desialylated bovine submaxillary-gland mucin. An increase in the rate of incorporation of sialic acid into desialylated glycoproteins was found after a lag period (7h) in regenerating liver. The increase was maximum 24h after partial hepatectomy for all acceptors tested. At later times after operation the sialyltransferase activity remained high only for desialylated fetuin. No soluble factors from liver or serum of partially hepatectomized animals influenced the activity of the sialyltransferases bound to the microsomal fraction. The sensitivity of sialyltransferases to activation by Triton X-100, added to the incubation medium, was unchanged in the microsomal preparation from animals 24h after sham operation or partial hepatectomy. The full activity of sialyltransferases towards the various desialylated acceptors showed some differences. Human Tamm–Horsfall glycoprotein was a good acceptor of sialic acid only when desialylated by mild acid hydrolysis. After this treatment, but not after enzymic hydrolysis, a decrease in molecular weight of human Tamm–Horsfall glycoprotein was observed. Further, the sialyltransferase activity as a function of incubation temperature gave different curves according to the acceptor used. The relationship between the biosynthesis of glycoproteins by regenerating liver and the sialyltransferase activity of microsomal fraction after partial hepatectomy is discussed.     

Possible involvement of DNA polymerases alpha and beta in bleomycin-induced unscheduled DNA synthesis in permeable HeLa cells

DNA polymerases involved in bleomycin-induced unscheduled DNA synthesis in some permeable human cells and rodent cells were studied by using selective inhibitors (aphidicolin, 2′,3′-dideoxythymidine-5′-triphosphate and N-ethylmaleimide) for DNA polymerases. The results suggest that both DNA polymerases α and β are involved in bleomycin-induced unscheduled DNA synthesis in permeable HeLa-S3 cells and probably in some other permeable human cells (HEp-2, KB and WI-38 VA-13 cells). Bleomycin-induced unscheduled DNA synthesis in some permeable rodent cells ($\text{SR-}\text{C3H}\text{He}$, $\text{Balb}\text{c}$ 3T3, 3Y1 and XC cells) is mostly attributed to DNA polymerase β.     

Difference in microviscosity induced by different cholesterol levels in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells

Microviscosity ($\text{}\text{?}$gh) in the surface membrane lipid layer of normal lymphocytes and malignant lymphoma cells, and in liposomes prepared from their lipid extracts, was determined with the aid of the fluorescence polarization properties of 1,6-diphenyl 1,3,5-hextriene embedded in it. The $\text{}\text{?}$gh values, both in intact cells and in the liposomes, are distinctively greater for normal lymphocytes than for the lymphoma cells, whereas the fusion activation energy in both types of cells and liposomes is 8 ± 0.5 kcal/mol. Determination of cholesterol revealed that its relative amount in a lymphoma cell is about half of that of a normal lymphocyte, a difference that may account for the above difference in fluidity. This thesis is supported by the observed changes in $\text{}\text{?}$gh, which follow artificial changes in cholesterol contents in the surface membrane of both cell types. Introduction of exogeneous cholesterol into the cell surface membranes was performed with lecithin-cholesterol (1:1) liposomes, and in lymphoma cells resulted in an increase of $\text{}\text{?}$gh to a level of normal lymphocytes. Extraction of native cholesterol from the cell surface membranes was carried out with lecithin liposomes, and in normal lymphocytes results in a decrease of $\text{}\text{?}$gh to a value similar to that of lymphoma cells. The induced changes in cholesterol contents are practically reversible for both cell types. By virtue of controlling the microviscosity of lipid layers, the level of cholesterol in cell surface membranes may play an important role in determining biological activities of normal and malignant cells.     

Alterations in labeling of cell-surface glycoproteins from normal and diabetic rat intestinal microvillous membranes

The effect of chronic streptozotocin-induced diabetes was studied on intestinal microvillous membrane surface carbohydrate groups. After 7 weeks of diabetes, purified microvillous membranes were prepared from rat small intestine and surface galactoproteins identified by labeling with galactose oxidase/sodium boro[³H]hydride. Membrane surface sialic acid residues were labeled using the sodium metaperiodate/sodium boro[³H]hydride technique. Membranes were solubilized in SDS and protein labeling analyzed by acrylamide electrophoresis. Membranes from diabetic rats showed an 81% increase in galactoprotein labeling ($\text{P< 0.02}$) while labeling of sialic acid residues was unchanged. The greatest increase in galactoprotein labeling occurred in protein monomers of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 116 000–200 000, where there was a 155% increase in labeling ($\text{P< 0.005}$). These results indicate that intestinal microvillous membrane protein glycosylation is altered in chronic diabetes. This increase in surface membrane carbohydrates could explain the decreased rates of proteolytic degradation previously described for at least one microvillous protein. An increase in membrane galactose groups has also been noted in hepatocyte and kidney glomerular basement membranes, which suggests the presence of a systematic change in membrane protein glycosylation occurring as a result of the diabetic state.     

Recognition of 2-keto-3-deoxyoctonate in bacterial cells and lipopolysaccharides by the sialic acid binding lectin from the horseshoe crab Carcinoscorpiusrotundacauda

The sialic acid binding loctin carcinoscorpin agglutinates $\text{Escharichia}\text{coli}\text{K12}\text{and}\text{Salmonella}\text{minnesots}$ R595 cells. This interaction can be inhibited by the saccharides namely 2-keto-3-deoxyoctonate and the disaccharide D-(N-acetylneuraminyl) (2→6)2-acetamide-2-deoxy-D-galactitol. N-acetylneuraminic acid is shown to be a poor inhibitor. The same behaviour is seen when purified lipopolysaccharides from these two Gram negative bacteria are used. $\text{Vibrio}\text{cholerae}$, a Grum negative bectarium devoid of 2-keto-3-deoxyoctonate and $\text{Staphylococcus}\text{sureus}$ a typical Gram positive bacterium failed to agglutinate in the presence of the lectin. The results suggest that the 2-keto-3-deoxyoctonate residues might represent the physiological substrate for the sialic acid binding lectin from the horseshoa crab.     

Biosynthesis in vitro and the pattern of lectin binding receptors in monkey kidney cell surfaces.

The glycosyltransferase activities involved in the biosynthesis $\text{in vitro}$ of neutral blood group-related glycosphingolipids were measured in African green monkey kidney cells (Vero) grown in culture. The a-fucosyltransferases which catalyzed the reaction between GDP-fucose and corresponding acceptors to form H-active and novel Le^a-type glycosphingolipids were characterized in membrane fractions isolated from Vero cells and monkey bone marrow. Using ¹²⁵I-labeled $\text{Ulex europeus}$ and $\text{Lotus tetragonolobus}$ lectins the differential binding to Vero cell surface glycoproteins and glycolipids was studied under various conditions.     

The serum sialyltransferase activity in alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin phenotypes Pi M and Z, purified by the thiol-disulfide exchange procedure, were desialylated by treatment with neuraminidase covalently coupled to Sepharose and used as acceptors of sialic acid in an assay system for serum sialic acid transferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. Both asialoantitrypsins were equally effective as acceptors in contrast to native Pi Z antitrypsin which did not accept any sialic acid. Serum sialyltransferase activity was determined in 38 adult alpha 1-antitrypsin deficient individuals (Pi Z, MZ, FZ, SZ) with normal liver function and was found to be of the same magnitude as the activity in normal individuals (Pi M). Equal activities were also found in 5 Pi Z patients with cirrhosis of the liver. The results strongly argue against the concept that sialyltransferase deficiency provides the molecular basis for alpha 1-antitrypsin deficiency.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

Cytotoxicity of pyocin S2 to tumor and normal cells and its interaction with cell surfaces

Cytotoxicity and adsorption of pyocin S2 produced by Pseudomonas aeruginosa M47 (PAO 3047) to virally transformed mammalian cells, human malignant cells and normal cells in the same species were studied. Pyocin S2 inhibited the growth of not only tumor cells (XC, TSV-5, mKS-A TU-7, HeLa-S3 and AS-II cells) but also normal cells (BALB/3T3 and BHK 21 cells). The inhibitory effects on the cells increased with an increase of pyocin S2 activity. On the other hand, there were some tumor cells (155-4 T2 and HGC-27 cells) and normal cells (normal rat kidney and human embryo lung cells) which were resistant to pyocin S2. The pyosin S2 activity was neutralized by the cell membrane preparations from pyosin S2-sensitive cells, but not by those from pyocin-resistant cells. This neutralization ability was inhibited by high concentrations of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine and}\text{N-}\text{acetyl}$ neuraminic acid and completely destroyed by periodate and neuraminidase. The inhibition by the saccharides was concentration dependent. These results suggest that the toxicity of pyocin S2 to several mammalian cells is due to the presence of the binding site for pyocin S2 in the cell membrane and further, that the carbohydrate moiety, especially of D-galactose, $\text{N-}\text{acetyl-}\text{D}\text{-galactosamine}$ and sialic acid, may play an important role as an initial binding site for pyocin S2.     

Surface carbohydrates of aged erythrocytes

Human erythrocytes, fractioned into populations of different density by ultracentrifugation in albumin gradients were examined to determine what changes in cell surface carbohydrates occur during their lifespan. In addition to changes occurring in $\text{N}$-acetylneuraminic acid ageing was accompanied by reduction in the $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and galactose content of erythrocyte membranes. These results show that extensive heterogeneity exists in the cell surface carbohydrate of the circulating population of erythrocytes and suggest clearance of neuraminidase treated erythrocytes may not be an adequate model for the removal of aged cells.     

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases,due to temperature dependence of kinetic parameters

Temperature dependence of asialomucin-sialyltransferase ($\text{CMP}\text{-N-}\text{acetylneuraminate:}\text{D}\text{-galactosyl-glyco-protein}$) N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in K_m and V_max values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.