Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells. Application of a micro-method for the semi-quantitation of surface-bound HRP

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competition between glycoprotein hormones and horseradish peroxidase for mannose-specific binding sites in cells of endocrine organs

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in paraformaldehyde-fixed, frozen sections of endocrine organs by a cytochemical method reported previously. In the testis, HRP was bound to interstitial cells, probably macrophages, and to sites extending along the surface of spermatozoa in the seminiferous tubules. In the epididymis, cells in the connective tissue, probably fibroblasts or macrophages, showed the specific reaction. In the ovaries, the reaction for lectin-bound HRP was observed in connective tissue cells of the theca externa, and in the mucosa of the uterus, binding of HRP occurred to many fibroblasts. The glycoprotein was also bound to cells in the connective tissue of the thyroid, probably mast cells, as well as to endothelial cells in the adrenal medulla and cortex. In all cases, the binding reaction required Ca2+ and was suppressed by mannose or mannan. Partially purified and highly purified preparations of glycoprotein hormones [ovine follicle-stimulating hormone, ovine luteinizing hormone, bovine thyroid-stimulating hormone, and human chorionic gonadotropin] as well as bovine thyroglobulin and yeast invertase competed with the binding of HRP to all the cells mentioned thus showing that the hormones were bound to the same sites as HRP. When 1 microM HRP was present in the incubation medium, the addition of 15-25 microM of highly purified hormones almost suppressed the reaction for lectin-bound HRP and competitive effects could be observed at even lower concentrations of the hormones.     

Mannose-specific binding sites for horseradish peroxidase in various cells of the rat

Mannose-specific binding sites for horseradish peroxidase (HRP) were studied in fixed sections of various tissues by a method reported previously. Liver sinusoidal cells, mast cells of lymph nodes, and alveolar macrophages of the lung and skin fibroblasts were main cell types showing mannose-specific binding of HRP. Macrophages, fibroblasts, and mast cells in the connective tissue of other organs also showed the reaction. However, macrophages of the spleen, and cultured 3T3 cells and L-cells did not give the reaction. The specificities of the binding reaction were studied by determining the approximate concentrations of competing sugars that suppressed the specific binding of HRP. It was found that the endogenous lectins in macrophages, fibroblasts, mast cells, and liver sinusoidal cells showed similar specificities toward various carbohydrates. D-Mannose and L-fucose had the highest affinity toward the lectins (competing ability for the binding of HRP). D-Mannose-6-phosphate, N-acetyl-D-glucosamine, D-glucose, D-ribose, and D-arabinose showed intermediate affinity, whereas D-xylose and D-galactose showed low affinity. Polymerized mannose in mannan and glycoproteins rich in mannose groups (invertase and ribonuclease B) showed much higher affinity to the binding sites than free mannose.     

Cytochemical observations on mannose-specific binding sites for horseradish peroxidase in liver sinusoidal cells

Paraformaldehyde-fixed, frozen sections of the liver of rats were processed for the detection of mannose-specific binding sites of horseradish peroxidase (HRP) by a method reported previously, with some modifications resulting in a more intense binding reaction. Before staining for peroxidase activity, the sections were held in buffered solutions of physiological saline at different temperatures and pH's, and in the presence or absence of added Ca2+, mannose or galactose. The gradual decrease and final disappearance of the binding reaction were observed. The release of HRP from the binding sites as determined by the disappearance of the cytochemical reaction was 50-100 times faster at 22 degrees C than at 4 degrees C and was 5-10 times faster at 37 degrees C than at 22 degrees C. The release was approximately twice as fast at pH 7.0 than at pH 9.0 and 20-30 times faster at pH 6.0 than at pH 7.0. The release of HRP was 10-15 times faster in the absence of 1 mM Ca2+ in the buffer solution and was approximately 100 times faster in the presence of 0.1 M D-mannose as compared to 0.1 M D-galactose. Pretreatment of the sections with trypsin abolished the binding reaction whereas neuraminidase, phospholipases A2 and C, and chondroitinase ABC were without effect. An acidic isoenzyme of HRP, Sigma type VIII, was bound more intensely and more widely to liver sinusoidal cells than another acidic isoenzyme, Sigma type VII, a basic isoenzyme, Sigma type IX, and the routinely used preparation, Sigma type VI. The effect of the temperature on the binding reaction was re-examined with an improved procedure. In contradistinction to the previous finding, strong binding of HRP after 2-4 h incubation at 4 degrees C was observed.     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells

Summary Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and postfixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6 phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding eaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and -lactalbumin were strong inhibitors. No addition of Ca²⁺ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H₂O₂-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, cLH, and hCG) did not do se. Addition of Ca²⁺ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calciam, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that -lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

Binding sites for horseradish peroxidase on the cell surface

Summary The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH₃ cells, and isolated rat liver cells was suppressed by 30 M monosialoganglioside, by 30 M trisialoganglioside, or by 5 mM CMP-neurminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyltransferase. The addition of 2 mM Mn²⁺ to the incubation medium with HRP suppressed the reaction for surfacebound HRP, and the addition of 10–20 mM Ca²⁺ intensified the reaction. The addition of 2 mM Zn²⁺ caused less inhibition than that of 2 mM Mn²⁺, and the addition of 2 mM Co²⁺ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

Morphology of leech retzius cells demonstrated by intracellular injection of horseradish peroxidase

• 1.1. The neuronal geometry of Retzius (R) cells in two species of leech (Hirudo medicinalis and Haemopis sanguisuga) was investigated by intracellular injection of horseradish peroxidase.
• 2.2. Each R cell sends major branches into the ipsilateral segmental nerves and, via the ipsilateral connectives, into the anterior and posterior adjacent ganglia.
• 3.3. No structural connection between the proximal axons of the two R cells could be detected, although numerous dendrites were demonstrated, some of which extended across the midline of the ganglion.
• 4.4. No major differences were found between the R cell morphology of the two species.

     

Characterization of the cryptogein binding sites on plant plasma membranes

Cryptogein is a 98-amino acid proteinaceous elicitor of tobacco defense reactions. Specific binding of cryptogein to high affinity binding sites on tobacco plasma membranes has been previously reported (K(d) = 2 nM; number of binding sites: 220 fmol/mg of protein). In this study, biochemical characterization of cryptogein binding sites reveals that they correspond to a plasma membrane glycoprotein(s) with an N-linked carbohydrate moiety, which is involved in cryptogein binding. Radiation inactivation experiments performed on tobacco plasma membrane preparations indicated that cryptogein bound specifically to a plasma membrane component with an apparent functional molecular mass of 193 kDa. Moreover, using the homobifunctional cross-linking reagent disuccinimidyl suberate and tobacco plasma membranes incubated with (125)I-cryptogein, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, two (125)I-cryptogein linked N-glycoproteins of about 162 and 50 kDa. Similar results were obtained using Arabidopsis thaliana and Acer pseudoplatanus plasma membrane preparations, whereas cryptogein did not induce any effects on the corresponding cell suspensions. These results suggest that either cryptogein binds to nonfunctional binding sites, homologues to those present in tobacco plasma membranes, or that a protein involved in signal transduction after cryptogein recognition is absent or inactive in both A. pseudoplatanus and A. thaliana.     

Unusual binding sites for horseradish peroxidase on the surface of cultured and isolated mammalian cells. Suppression of binding by certain nucleotides and glycoproteins, and a role for calcium

Binding sites for horseradish peroxidase (HRP), with unusual properties, were detected on the surface of cultured and isolated cells after the cells (on cover slips) had been quickly dried, fixed in cold methanol, and post-fixed in a paraformaldehyde solution. The reaction for surface-bound HRP was suppressed by micromolar concentrations of glycoproteins such as invertase, equine luteinizing hormone (eLH) or human chorionic gonadotropin (hCG). The reaction was also suppressed by 20 mM CDP, UDP, GTP, NAD, and ribose 5-phosphate. Two to six times higher concentrations of GMP, fructose 1-phosphate, galactose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, and glucose 6-phosphate were required to suppress the binding reaction. AMP, ATP, heparin, mannan, and eight non-phosphorylated sugars showed relatively low competing potencies but fucoidin and alpha-lactalbumin were strong inhibitors. No addition of Ca2+ was required for the binding of HRP to the cell surface. However, calcium-depleted, inactive HRP did not compete with the binding of native (calcium-containing) HRP whereas H2O2-inactivated HRP suppressed the binding. GTP, NAD, ribose 5-phosphate, and EGTA accelerated the release of previously-bound HRP from the cell surface whereas glycoproteins (invertase, eLH, and hCG) did not do so. Addition of Ca2+ to GTP, NAD, ribose 5-phosphate or to EGTA prevented the accelerated release of HRP from the cell surface. It is suggested that calcium, present either in the surface membrane or in HRP itself, is involved in the binding of HRP to the cell surface and in the inhibition of binding by GTP, NAD, and ribose 5-phosphate. It is also suggested that alpha-lactalbumin, GTP, UDP, and CDP compete with the binding of HRP to a glycosyltransferase on the cell surface.     

A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.     

Binding sites for horseradish peroxidase on the cell surface. Suppression of binding by gangliosides and effects of some bivalent cations

The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH3 cells, and isolated rat liver cells was suppressed by 30 microM monosialoganglioside, by 30 microM trisialoganglioside, or by 5 mM CMP-neuraminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyl-transferase. The addition of 2 mM Mn2+ to the incubation medium with HRP suppressed the reaction for surface-bound HRP, and the addition of 10-20 mM Ca2+ intensified the reaction. The addition of 2 mM Zn2+ caused less inhibition than that of 2 mM Mn2+, and the addition of 2 mM Co2+ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

Competition between histone H1 and HMGN proteins for chromatin binding sites

The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higher-order chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1–GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.     

Enhanced number of actin binding sites on plasma membranes of polyoma virus-transformed fibroblasts

Plasma membranes, isolated from normal (C13) and polyoma virus-transformed (J1) cultured BHK cells were incubated with G-actin under polymerizing conditions, followed by a low-speed centrifugation. The amount of actin attached to the pelleted BHK-J1 plasma membranes was at least twice that on BHK-C13 membranes, indicating a greater number of actin attachment sites on the former. This result was confirmed by the observation that the plasma membranes from the transformed cells were also more active in nucleating polymerization of pyrene-labelled actin. Most of the actin attachment sites could be solubilized by Triton or low-salt extraction treatment.     

Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes

Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.     

Lipid A binding sites in membranes of macrophage tumor cells

Lipopolysaccharide affects a variety of eukaryotic cells and mammalian organisms. These actions are involved in the pathogenesis of Gram-negative septicemia. Many of the actions of lipopolysaccharide are believed to be caused by its active moiety, lipid A. Our laboratory has previously identified a bioactive lipid A precursor, termed lipid IVA (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16888), which can be labeled with 32P of high specific activity and purified. In this work we have used the labeled probe, 4'-32P-lipid IVA, to develop a novel assay for the specific binding of lipid IVA to whole cells. We have also demonstrated its use in a ligand blotting assay of immobilized cellular proteins. Using the whole cell assay, we show that 4'-32P-lipid IVA specifically binds to RAW 264.7 macrophage-like cultured cells. The binding is saturable, is inhibited with excess unlabeled lipid IVA, and is proteinase K-sensitive. It displays cellular and pharmacological specificity. Using the ligand blotting assay, we show that several RAW 264.7 cell proteins can bind 4'-32P-lipid IVA. The two principal binding proteins have Mr values of 31 and 95 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractionation studies indicate that the 31-kDa protein is enriched in the nuclear fraction and may be a histone, whereas the 95-kDa protein is enriched in the membrane fraction. The binding assays that we have developed should lead to a clearer understanding of lipid A/animal cell interactions.     

High affinity thyroid hormone binding sites on purified rat liver plasma membranes.

Two orders of saturable binding sites for L-T₃ were detected on purified rat liver plasma membranes--a high affinity, low capacity binding site with a Kd of 3.2 ± 0.5 nM, and a lower affinity, higher capacity site with a Kd of 220 ± 50 nM. Competition-inhibition studies revealed that both D-T₃ and L-T₄ (two compounds with lower biological potencies than L-T₃) were also less potent than L-T₃ in competing for these binding sites. The present studies demonstrate, therefore, the presence of specific thyroid hormone binding sites on rat liver plasma membranes. In addition, they suggest that these sites may have a role both in mediating the known effects of thyroid hormones on membrane functions, and in regulating the entry of thyroid hormones into target cells.