Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: concept of a leading edge.

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with [meso-3H]diaminopimelic acid ([3H]Dap). The second method was autoradiography of cells pulse-labeled with [3H]Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surface components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of [3H]Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific beta-lactam antibiotic furazlocillin did not affect [3H]Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Partial Purification and Some Properties of Aggregation Factor from Pronase-Susceptible Floe Forming Flavobacterium Strain B

Globomycin inhibited the incorporation of [¹⁴C]diaminopimeric acid (Dap) into the cold 5% TCA insoluble fraction of Escherichia coli H2143 at higher concentrations than the minimum inhibitory concentration (MIC).

One-sixth or -seventh molecules of the lipoprotein were found per one molecule of N-acetyl glucosamine (GlucNAc) or Dap in globomycin-treated cells as compared with one-twelfth or -thirteenth molecules in normal cells. Among globomycin-resistant cells isolated, one-tenth were lipoprotein-less mutants and they showed a slightly swollen form and leaked RNase into the medium. It was interesting that spheroplast formation of the mutants in the presence of the antibiotic was not observed even at a high concentration.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

125I]EGF binding ability is stable throughout the replicative life-span of WI-38 cells

Using a serum-free medium supplemented with hormones and growth factors, which included epidermal growth factor (EGF), we investigated the binding and processing-degradation of [125I]EGF in WI-38 cells of various in vitro ages. The binding and processing-degradation systems of these cells remained essentially unchanged throughout their lifespan. The number of specific [125I]EGF binding sites per cell increased as the cultures senesced, though the number of specific binding sites per micron 2 (surface area) remained constant. The kinetics of ligand degradation as well as the qualitative and quantitative nature of the degradation products also remained essentially unchanged throughout the life-span. The only consistent alteration in any of the binding parameters measured was the slight decrease in the apparent Kd of the ligand-receptor complex, independent of temperature. Quantitation of EGF-stimulated DNA synthesis revealed a decrease in the percentage of cells incorporating [3H]thymidine ([3H]TdR) during a 30-h exposure from 45% in young cells to 0.25% in senescent cells, although [125I]EGF binding or processing-degradation did not differ significantly in young and old cells. Thus, EGF binding does not decrease in senescence.     

Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by forskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of [3H]-forskolin (10nM) to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of [3H]-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC50 value of 3 x 10(-7) M. Results showed that the number of [3H]-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC50 values for inhibition by unlabeled forskolin of binding of [3H]-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.     

Determination of murein precursors during the cell cycle of Escherichia coli

A convenient and reliable method has been established that allows a quantitative determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography the lipid-linked intermediates were hydrolysed to release the muropeptides. The accuracy for the measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was +/- 1.9% (SD), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) +/- 10% (SD) and for undecaprenyl-P-P-(GlcNAc-beta 1----4)MurNAc-pentapeptide (lipid II) +/- 5% (SD). The ratio of UDP-MurNAc-pentapeptide:lipid I:lipid II was about 300:1:3 for E. coli MC4100. The relative cellular concentrations of all three precursor molecules were found not to vary throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is not controlled by oscillations in the concentrations of these late murein precursors.     

Stoichiometry of receptor-Gs-adenylate cyclase interactions.

Little is known about the relative stoichiometry of guanine nucleotide-binding (G) proteins relative to the effector systems to which they link. We addressed this question for the stimulatory G protein (Gs) linked to adenylate cyclase. Forskolin stimulates the catalytic subunit of adenylate cyclase (C), but it has a higher efficacy and potency when C also interacts with the G protein Gs. Accordingly, we measured high-affinity [3H]forskolin binding to intact cells to assay alpha s-C complexes. No high-affinity specific binding occurred with unstimulated cells. The beta-adrenergic agonist isoproterenol promoted the binding of [3H]forskolin to about 3000 sites per cell, suggesting that each receptor on average activates at least several Gs molecules. Activating Gs directly with cholera toxin maximally promoted [3H]forskolin binding to a similar number of sites, suggesting that this is the maximal number of alpha s-C complexes formed per cell. We conclude that each cell likely contains only a few thousand functional copies of C, and that the availability of C (rather than Gs, which exists in more than 100,000 copies per cell) is likely to be limiting for agonist stimulation of adenylate cyclase activity.     

Measurement of proviral genes in uninfected and avian myeloblastosis virus-infected cells by hybridization with 3H-labeled complementary DNA probe excess.

Viral RNA (vRNA) from avian myeloblastosis virus or DNA from virus-infected and uninfected cells was hybridized with [3H]DNA complementary to viral RNA ([3H]cDNA) under conditions of [3H]cDNA excess. When [3H]cDNA was used to drive the hybridization reaction with vRNA, a rate constant of 33.2 liters/mol-s was obtained. The same rate constant was obtained when vRNA excess was used as the driver. The specific activities of the [3H]DNA probe, estimated from kinetic measurements of the hybridization reaction and from the amount of [3H]cDNA in hybrid form at equilibrium, were 9.1 and 8.6 cpm/pg, respectively. DNA isolated from uninfected cells contained five or six copies of proviral DNA per cell genome. DNA isolated from erythrocytes infected with avian myeloblastosis virus had an additional five or six viral genes added to the cell genome, and the virus-infected target cell (myeloblasts) contained about 15 additional copies of proviral DNA per cell. The use of excess [3H]cDNA probe is an easy and accurate method to quantify the frequency of proviral DNA sequences in cell DNA and to measure a small amount (40 to 200 pg) of vRNA. Probe excess hybridization offers a number of advantages over other procedures and these are discussed.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

A colloidal gold labeling technique for the direct determination of the surface area of eukaryotic cells

We have developed a colloidal gold labeling technique for the direct quantitation of the cell surface area. The method is based on coating the cell surface with [195Au] colloidal gold-protein complexes followed by morphometric determination of the labeling density (gold particles/micron2 cell surface) and radiometric determination of the total number of gold particles bound per cell. The ratio of both values directly gives the cell surface area. The accuracy of the method was shown using Staphylococcus aureus cells as a model system, where the cell surface area determined with our assay (4.0 microns2) corresponded well to the value calculated from the radius of the cells (3.6 microns2). In a more complex model system J-774 mouse macrophages were labeled with different amounts of [195Au] gold-protein complexes to show that the assay is independent of the degree of saturation of the cell surface binding sites. Both high (135 Au/microns2) and low (65 Au/microns2) labeling densities resulted in a surface area of about 1200 microns2. The technique finally was applied to L-929 fibroblasts to determine the increase of the cell surface area when the cells change from a spherical to a flat monolayer state. We found that the cell surface area increased 3-fold during the spreading process. The results show that the colloidal gold labeling technique allows the direct determination of the surface area of complex eukaryotic cells. The technique is suitable for the quantitation of changes in the surface architecture known to occur in different functional states of eukaryotic cells.     

Incorporation of antigen 125I IgG into particular cell compartments: binding by chromatin

Incorporation of [125I]IgG into spleen cells was studied in vivo and in vitro. In vivo, the antigen after uptake into the cytoplasm migrated into cell nuclei, where it was bound to chromatin up to the saturation level. One day after immunization the constant level of [125I]IgG was 1.3 X 10(12) molecules per spleen (10(8) cells). The same number of [125I]IgG molecules were bound to chromatin in cell cultures. The uptake of [125I]IgG was competitively inhibited by non-labelled IgG. Binding of [125I]IgG molecules reextracted from cytoplasm and chromatin with specific anti-human IgG serum argues against the uptake of degraded [125I]IgG molecules. [125I]IgG was tightly bound to DNA. Approximately 50 per cent of [125I]IgG was present in the residual chromatin fraction (after removal of 0.35 M and 2 M NaCl-soluble fractions) and 40 per cent was complexed with DNA (after removal of histones and non-histones AP1, AP2, AP3 and AP4). Binding of [125I]IgG by isolated chromatin was inhibited by the cytoplasmic fraction but not by BSA. Binding of [125I]IgG by fractionated chromatin, (chromatins remaining after removal of 0.35M, and 2M NaCl-soluble fractions or histones + non-histones AP1 + AP2 + AP3 + AP4) occurred at a level similar to that observed with native chromatin. The results suggest that interaction of antigen with immunocompetent cells is not restricted to the cell surface but that antigen seems to be taken up into cytoplasm, migrates to the nuclei and is bound to chromatin, probably directly to DNA. The results are discussed in relation to the induction of the immune reaction.     

Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.     

Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

We have documented a single, specific binding site for [3H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes. The binding of the radioligand to its receptor is reversible with cold H1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), we calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity (mean KD +/- SD; 3.8 +/- 4.8 nM) for [3H]pyrilamine, followed by T helper cells (KD = 5.0 +/- 6.6 nM), B cells (KD = 14.2 +/- 2.0 nM), and T suppressor cells (KD = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H1 receptors per cell (35,697 +/- 15,468), followed by B cells (10,732 +/- 9060), T helper cells (6838 +/- 8167), and monocytes (5589 +/- 2266). The kinetics of binding for this radioligand was carried out in resting and mitogen-stimulated T cells over a 48-hr period. We found that the binding affinity for [3H]pyrilamine increased over the 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [3H]pyrilamine decreased over the 48-hr period. Preincubation of T cells with unlabeled histamine before carrying out the radioligand binding assay resulted in a decrease in the binding affinity of the receptors to [3H]pyrilamine, but the number of receptors per cell did not change significantly. Although the function of H1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in modulating the immune response.     

Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.

Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.     

Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.