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G M Edelman

Publications and source records attributed to G M Edelman.

At least 217 records · Page 12Linked to original sources
PubMed1983

Neuron-glia adhesion is inhibited by antibodies to neural determinants.

Suspensions of embryonic chick neuronal cells adhered to monolayers of glial cells, but few neurons bound to control monolayers of fibroblastic cells from meninges or skin. Neuronal cell-glial cell adhesion was inhibited by prior incubation of the neurons with Fab' fragments of antibodies to neuronal membranes. In contrast, antibodies to the neural cell adhesion molecule (N-CAM) did not inhibit the binding. These results suggest that a specific adhesive mechanism between neurons and glial cells exists and that it is mediated by CAM's that differ from those so far identified.

Animals↗
PubMed1983

Comparison of the 34,000-Da pp60src substrate and a 38,000-Da phosphoprotein identified by monoclonal antibodies.

One of the cellular targets of the pp60src tyrosine kinase is a phosphoprotein with a Mr = 34,000 and an isoelectric point of approximately 7.5 (Radke, K., Gilmore, T., and Martin, G. S. (1980) Cell 21, 821-828; Erikson, E., and Erikson, R. L. (1980) Cell 21, 829-836). We report here the preparation of monoclonal antibodies to partially purified 34-kDa protein and to a heretofore unrecognized phosphoprotein that is not a pp60src target. Two antibodies were initially obtained that recognized phosphoproteins in the Mr = 34,000-39,000 range. One of these antibodies immunoprecipitated a 34,000-Da protein which, on the basis of its molecular mass, phosphorylation state, and isoelectric point, was determined to be the 34-kDa pp60src substrate. The second monoclonal antibody bound to a 38,000-Da nucleolar associated protein, which appeared not to be a target of the pp60src kinase and was found by tryptic analysis to be structurally unrelated to the 34-kDa protein. The monoclonal antibody to the 34-kDa protein coupled to Sepharose CL-4B was used to purify the pp60src substrate to homogeneity in milligram quantities. Both the purified 34-kDa protein and the monoclonal antibody are currently being used in studies aimed at elucidating the structure and function of this pp60src target.

Animals↗
PubMed1983

Cell adhesion molecules.

It has been proposed that cell-cell recognition occurs by means of local cell surface modulation of a small number of proteins rather than by expression of large numbers of different cell surface markers. Several different cell adhesion molecules (CAM's) have now been found in a number of vertebrate species in different tissues such as liver and striated muscle and even in a single complex structure such as the brain, where different molecules specific for neurons and glia have been identified. The neuron-specific molecule is involved in early embryonic events but also mediates neurite fasciculation, neuromuscular interaction, and orderly layering of neural tissue. It undergoes local surface modulation with loss of sialic acid during development. A failure of this process is closely correlated with connectional disorders in the staggerer mutant of the mouse. The accumulated data on this and other CAM's favor modulation theories rather than strict chemoaffinity theories of cell-cell recognition.

Animals↗
PubMed1983

The 34 kd pp60src substrate is located at the inner face of the plasma membrane.

The subcellular localization of the 34 kd protein substrate of the pp60src kinase was investigated by immunofluorescence microscopy and subcellular fractionation. When permeabilized fibroblasts were stained with a monoclonal anti-34 kd protein antibody, a diffuse reticular pattern was observed. The 34 kd protein was not exposed on the outside surface of the cell. Double immunofluorescence staining experiments established that the 34 kd protein distribution was similar to that of the membrane-associated protein alpha-spectrin. The 34 kd protein was found in cell sections to be concentrated along the cell edges. Taken together, these results suggested that the 34 kd pp60src substrate was associated with the inside surface of the plasma membrane. This conclusion was supported by subcellular fractionation experiments in which the 34 kd protein was observed to fractionate with the plasma membrane. These localization studies support further the hypothesis that many of the primary effects of the pp60src kinase occur at the plasma membrane.

Antibodies, Monoclonal↗
PubMed1983

Molecular topography of the neural cell adhesion molecule N-CAM: surface orientation and location of sialic acid-rich and binding regions.

Chemical analyses and binding studies have been correlated to clarify the relationship of structure to function in the neural cell adhesion molecule (N-CAM) from embryonic chicken brain. N-CAM isolated from the cell surface appears to include two closely related polypeptide chains. Treatment with neuraminidase of such preparations of N-CAM bound by antibodies on solid supports yielded components of Mr 140,000 and 170,000. These components each had the same amino-terminal sequence as N-CAM and gave nearly identical profiles on peptide maps. Immunoprecipitation of N-CAM from 9-day brain cells treated with tunicamycin yielded corresponding components of Mr 130,000 and 160,000, suggesting that the differences between these two components of N-CAM are in the polypeptide rather than the carbohydrate portions of the molecules. N-CAM appears to be oriented with the amino terminus extending away from the cell surface and with the bulk of the sialic acid near the middle of the peptide chain. As shown previously, incubation of N-CAM at 37 degrees C generates a fragment (Fr1) of Mr 65,000 that lacks most of the sialic acid. Treatment of membranes with Staphylococcus aureus V-8 protease released a fragment (Fr2) of N-CAM that contained most of the sialic acid; this fragment had an Mr of 108,000 after neuraminidase treatment. Both of these fragments contain the amino-terminal portion of the polypeptide chain. At least a portion of the N-CAM binding site was found to be located in the amino-terminal region of the peptide chain. Most or all of the sialic acid was not directly involved in binding, although it can influence binding, as indicated by the finding that neuraminidase-treated N-CAM (desialylated-N-CAM) bound to cells to a greater extent than untreated N-CAM. The Fr1 and the Fr2 fragments in solution did not bind to cells but were as effective as N-CAM and desialylated-N-CAM as competitors for N-CAM binding to cells. When fixed covalently to beads, N-CAM, desialylated-N-CAM, and the Fr1 and Fr2 fragments bound specifically to cells. In contrast, the N-CAM autolysis products released along with Fr1 neither bound to cells nor competed for N-CAM binding. In addition to suggesting a location for the N-CAM binding region, the accumulated results raise the possibility that valence may play a key role in N-CAM binding.

Animals↗
PubMed1983

Early epochal maps of two different cell adhesion molecules.

N-CAM, the neural cell-adhesion molecule, has previously been found to be expressed during several epochs of development and function, first as an early marker in embryo-genesis, later during organogenesis, and finally in adult life. L-CAM, the liver cell-adhesion molecule, has now been localized in embryonic and adult tissues of the chicken by fluorescent antibody techniques. In the early embryonic epoch, L-CAM and N-CAM appeared in epiblastic and hypoblastic tissues. L-CAM was distributed thereafter across all three germ layers. By the onset of neurulation, however, L-CAM disappeared in the region of the neural plate and N-CAM increased in amount in that region. L-CAM appeared strongly on all budding endodermal structures (liver, pancreas, lung, thyroid, parathyroid, thymus, and bursa of Fabricius) whereas N-CAM appeared most strongly in the neural plate, neural tube, and in cardiac mesoderm but was not found in endodermal derivatives. In placodes, both L-CAM and N-CAM were present until the formation of definitive neural structures, at which time L-CAM disappeared. In kidney precursors, the two CAMs followed a complex reciprocal pattern of appearance and disappearance. For the most part, however, the distributions of the two molecules did not overlap during organogenesis. Like N-CAM, L-CAM persisted in a distinctive pattern of expression in adult tissues. During embryonic development, the two different CAMs were distributed on tissues derived from more than two-thirds of the early embryonic surface. Interpretation of maps summarizing CAM distributions over a defined developmental epoch suggested a key role for both L-CAM and N-CAM in embryonic induction. Consistent with this interpretation and with the fact that the continuity of germ layers is lost when organ rudiments are formed, neither of the CAMs was limited in distribution to a single germ layer. The regions of the early epochal maps that lacked both L-CAM and N-CAM comprised some portions of the splanchnopleure and somatopleure. Certain adult tissues that derive from this lateral plate mesoderm such as smooth muscle also lacked L-CAM and N-CAM. Such observations suggest that at least one more CAM may exist in these and similarly derived tissues.

Animals↗
PubMed1983

Kinetics of homophilic binding by embryonic and adult forms of the neural cell adhesion molecule.

The neural cell adhesion molecule, N-CAM, is a cell surface glycoprotein found on embryonic and adult neurons and on a variety of ectodermal and mesodermal tissues in very early embryos. During development, it shows local variations in prevalence at the cell surface as well as conversion from an embryonic form (E form) with high sialic acid content to an adult form (A form) with lesser amounts of this sugar. This E leads to A conversion occurs on different schedules in different brain regions, and it has been hypothesized that both the conversion and the prevalence changes are related to early regulation of pattern formation and connectivity. In order to identify precisely the consequences of these mechanisms of local cell surface modulation of N-CAM, an assay was developed to measure the rate of aggregation either of vesicles reconstituted from lipid and purified N-CAM or of native brain membrane vesicles. In both preparations, aggregation was greater than 95% inhibitable by specific anti-(N-CAM) Fab' fragments. The rates of aggregation of reconstituted N-CAM vesicles and native brain vesicles were found to be inversely related to the sialic acid content of their N-CAM molecules, with full desialylation resulting in about a 4-fold increase in rate over E-form N-CAM. Intermediate rates were obtained both with A-form N-CAM (which contains only one-third of the sialic acid content of E-form N-CAM) and with partially desialylated E-form N-CAM. The rate of coaggregation of reconstituted vesicles containing E-form N-CAM with reconstituted vesicles containing A-form N-CAM was also intermediate, implying that desialylation did not change the nature of (N-CAM)-(N-CAM) binding but only its rate. Even larger alterations in vesicle aggregation rate were seen when the amount of N-CAM per vesicle was altered. A 2-fold increase in the N-CAM-to-lipid ratio of reconstituted vesicles resulted in a greater than 30-fold increase in their rate of aggregation. Moreover, desialylation did not cause a further increase in the rate of aggregation of these already rapidly aggregating vesicles. These results in a model system demonstrate the large range of binding rates that are obtainable by various forms of local surface modulation of N-CAM. They are consistent with the proposal that similar alterations affecting (N-CAM)-mediated cell adhesion in vivo may be major factors in pattern formation during development of the nervous system.

Age Factors↗
PubMed1983

Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells.

We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of M(r) 81,000 on NaDodSO(4)/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the M(r) 81,000 species that had been further purified on NaDodSO(4)/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the M(r) 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major M(r) 124,000 component, with minor components of M(r) 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of M(r) 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of M(r) 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of M(r) 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble M(r) 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.

Antibodies, Monoclonal↗
PubMed1983

Neural cell adhesion molecule mediates initial interactions between spinal cord neurons and muscle cells in culture.

Previous studies in this laboratory have described a cell surface glycoprotein, called neural cell adhesion molecule or N-CAM, that appears to be a ligand in the adhesion between neural membranes. N-CAM antigenic determinants were also shown to be present on embryonic muscle and an N-CAM-dependent adhesion was demonstrated between retinal cell membranes and muscle cells in short-term assays. The present studies indicate that these antigenic determinants are associated with the N-CAM polypeptide, and that rapid adhesion mediated by this molecule occurs between spinal cord membranes and muscle cells. Detailed examination of the effects of anti-(N-CAM) Fab' fragments in cultures of spinal cord with skeletal muscle showed that the Fab' fragments specifically block adhesion of spinal cord neurites and cells to myotubes. The Fab' did not affect binding of neurites to fibroblasts and collagen substrate, and did not alter myotube morphology. These results indicate that N-CAM adhesion is essential for the in vitro establishment of physical associations between nerve and muscle, and suggest that binding involving N-CAM may be an important early step in synaptogenesis.

Animals↗
PubMed1983

A kinetic study of the oxidation by molecular oxygen of the cytochrome chain of intact yeast cells, Acetobacter suboxydans cells, and of particulate suspensions of heart muscle.

The pre-steady state kinetics of the cytochrome c oxidase reaction with oxygen were studied by a variation in the reaction time between approximately 6 and 25 ms at oxygen concentrations less than 6 mumol/l. For baker's yeast, a pseudo-first-order velocity constant of approximately 150 s-1 at 1.3 mumol/l O2 was obtained corresponding to a second-order reaction between O2 and a3 at a forward velocity constant (k+1) of approximately 3 X 10(7) liter equiv.-1s-1. Thus, the membrane-bound oxidase in the intact cell exhibits one of the most rapid enzyme-substrate reactions to be reported. The value is identical with that of Greenwood and Gibson on an isolated, solubilized cytochrome c oxidase. Similar values of k+1 are calculated from the turnover numbers [k+2 (a+2)] divided by the Km values (formula; see text) measured for these yeast preparations, which points to an almost negligible reverse reaction (k-1) compared to k+2(a+2). Similar calculations for the membrane-bound cytochrome c oxidase of heart muscle give a value of k+1 approximately equal to 10(7) liter equiv.-1s-1. The concordance of the different values of k+1 supports the view that the yeast cell wall does not impart a significant diffusion barrier to the transport of molecular oxygen. In contrast, Acetobacter suboxydans exhibits a much larger value for Km, and has a terminal oxidase of different kinetic parameters.

Acetobacter↗
PubMed1982

Chemical characterization of a neural cell adhesion molecule purified from embryonic brain membranes.

A neural cell adhesion molecule (N-CAM) was purified in milligram quantities from detergent extracts of embryonic chick brain membranes. N-CAM has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. The carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms. N-CAM appears to be an integral membrane protein in that its extraction from membranes required detergent. Although soluble, the purified molecule was aggregated (Mr = 0.5 to 1.2 X 10(6)) and polydisperse in detergent-free solutions. N-CAM from brain also migrated as a broad but continuously stained region from Mr = 200,000 to Mr = 250,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecule from retina was similar but had a somewhat faster mobility. Desialation of N-CAM did not significantly change its behavior in solution, but converted both brain and retinal N-CAM to components migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as material of about Mr = 140,000. Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain. On prolonged incubation at neutral pH, N-CAM undergoes apparent proteolysis to yield a polypeptide that contains little sialic acid and has a Mr = 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a separate sialic acid-rich component, and a variety of small peptides. The 65,000-dalton polypeptide appeared to contain all of the antigenic determinants of intact N-CAM that neutralize the adhesion-blocking ability of anti-retinal cell Fab' fragments.

Amino Acid Sequence↗
PubMed1982

Protein complexes from active replicative fractions associate in vitro with the replication origins of yeast 2-micrometers DNA plasmid.

In a search for a replication complex, the activity that replicates the 2-micrometers yeast DNA plasmid in vitro was isolated in a high molecular weight form (Mr approximately 2 X 10(6) by gel filtration and rate-zonal sedimentation from extracts prepared from cells of the budding yeast Saccharomyces. When obtained from cells in late logarithmic cultures this material or "complex" was labile compared to that from early logarithmic cultures, and it did not survive as a complex after ammonium sulfate precipitation. This suggests that, as cultures approach stationary phase and cells cease growth, the association of its protein constituents may be altered. A chimera of 2-micrometers DNA inserted into the plasmid pBR322 was used to test for binding of components of the complex. After a brief incubation of the chimera in vitro with the high molecular weight material containing replicating activity, a protein "knob" was found associated with the 2-micrometers DNA as shown by electron microscopy. This association was not random but was limited to two positions on the plasmid. In the same series of experiments, the in vitro origins of 2-micrometers plasmid replication were also mapped. Two origins were found, consistent in position with those that have been identified in vivo. Molecules utilizing both origins simultaneously in vitro were not observed, and replication in vitro was bidirectional. The location of the origins corresponded to the positions at which the protein knobs associated with 2-micrometers DNA. This and the fact that no replicative intermediates with associated complexes were detected raises the possibility that a specific protein complex may be involved in initiation of DNA replication.

DNA Replication↗
PubMed1982

Neural cell adhesion molecules in rodent brains isolated by monoclonal antibodies with cross-species reactivity.

Previous studies in this laboratory have led to the identification and purification of a chicken cell surface protein named "neural cell adhesion molecule" (N-CAM) that is involved in neural cell-cell and neurite-neurite interactions. In the present investigation, we have found that a similar molecule exists in the mouse and have confirmed that it is also present in rat neural tissue. A monoclonal antibody to chicken N-CAM that crossreacted with mouse and rat brains and an independently derived monoclonal antibody to mouse N-CAM were used to purify an antigen from perinatal mouse and rat brains. The purified neural antigen resembles chicken N-CAM in its ability to neutralize antibodies that inhibit neural cell aggregation and also in its biochemical properties including molecular weight, sialic acid content, amino acid composition, and autoconversion to a smaller polypeptide. Like chicken N-CAM, the murine molecule is found throughout the nervous system and over the entire neuronal cell surface. These results strongly suggest that the molecule is evolutionarily related to chicken N-CAM and prompt the hypothesis that cell adhesion involving N-CAM is a fundamental mechanism existing in nervous systems of different phylogenetic classes of animals.

Aging↗
PubMed1982

Binding properties of a cell adhesion molecule from neural tissue.

We have previously identified and purified a cell surface glycoprotein from retina and brain, called neural cell adhesion molecule or N-CAM, that appears to be involved in neural cell--cell adhesion, the fasciculation of neurites, and the formation of normal tissue patterns in the retina. The present studies reveal that artificial vesicles containing lipid and purified N-CAM bind to different cell types with a specificity similar to that of nerve cells. The same results were obtained with soluble N-CAM that had been briefly exposed to pH 3. In both cases the binding altered the rate of aggregation of neural cells and, like cell--cell adhesion, was inhibited by antibodies against N-CAM. The results support the proposal that N-CAM is a ligand in the formation of bonds between nerve cell membranes. Moreover, results of studies of vesicle--vesicle interactions and of N-CAM binding to cells coated with anti-(N-CAM) Fab' fragments were consistent with the idea that the N-CAM molecules on different cells may interact directly to form cell--cell bonds.

Animals↗