After internalization of 125I-asialo-orosomucoid (ASOR) by isolated rat hepatocytes, ligand dissociates by two kinetically distinct pathways (Oka and Weigel, J. Biol. Chem. 257, 10,253, 1983). These ...slow and fast dissociation pathways correspond to two functionally different subpopulations of cell surface galactosyl receptors designated, respectively, State 1 and State 2 receptors. Freshly isolated cells or cells equilibrated below 24 degrees C express only State 1 receptors. Cells equilibrated at 37 degrees C express both State 1 and State 2 receptors. Ligand dissociation after internalization of surface-bound 125I-ASOR was measured using the permeabilizing detergent, digitonin. The slow dissociation pathway was mediated by State 1 receptors and was the only pathway expressed by cells which were freshly isolated or had been equilibrated at 24 degrees C. State 2 receptors are expressed at temperatures above about 20 degrees C, and both the fast and slow dissociation pathways occurred in cells equilibrated at 37 degrees C. State 2 receptors therefore mediate the rapid dissociation pathway. Dissociation and subsequent degradation of specifically bound ligand routed in either pathway were complete, respectively, within 3 and 6 hrs.
Intact isolated rat hepatocytes show a small amount of specific
125I-labeled hyaluronic acid (HA) binding. However, in the presence of digitonin, a very large increase in the specific binding of
...125I-HA is observed. Chondroitin sulfate, heparin and dextran sulfate were as effective as unlabeled HA in competing for
125I-HA binding to permeabilized hepatocytes, indicating that the binding sites may have a general specificity for glycosaminoglycans. After rat hepatocytes had been homogenized in a hypotonic buffer, more than 98% of the
125I-HA binding activity could be pelleted by centrifugation at 100 000 × g for 1 h. Mild alkaline treatment of hepatocyte membranes did not release
125I-HA binding activity, suggesting that the HA binding site is an integral membrane molecule. Furthermore, trypsin treatment of deoxycholate-extracted membranes destroyed the binding activity, as assessed by a dot-blot assay. This suggests that a protein component in the membrane is necessary for
125I-HA binding activity. Rat fibrinogen could be a possible candidate for the HA binding activity because HA binds specifically to human fibrinogen (LeBoeuf et al. (1986) J. Biol. Chem. 261, 12 586). Also, fibrinogen can be found in a quasi-crystalline form in rat hepatocytes and could be pelleted with the membranes. Rat fibrinogen was not responsible for the
125I-HA binding activity, since (1) purified rat fibrinogen did not bind to
125I-HA, and (2) immunoprecipitation of rat fibrinogen from hepatocyte extracts did not decrease the
125I-HA binding of these extracts. We conclude that the internal HA binding sites are membrane- or cytoskeleton-associated proteins and are neither cytosolic proteins nor fibrinogen.
The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. ...To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total 125I-asialo-orosomucoid (125I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both 125I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the 125I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.
Virtually all of the known enzymes utilize one, two, or three substrates to produce one or two products. The hyaluronan (HA) synthase enzyme family is very unusual, and perhaps unique, in that these ...synthases have two different enzymatic activities within the same protein. Furthermore, the growing HA chain produced after each sugar addition becomes the substrate for the next sugar addition. HA synthases are also membrane-bound enzymes. In bacteria and eukaryotic cells, active HA synthase is localized to the plasma membrane. The two activated sugar precursors for HA biosynthesis are UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). In order to make a disaccharide unit and extend the growing HA chain, HA synthase possesses at least six distinct functions. These activities include two different sugar nucleotide binding sites, two different glycosyltransferase activities, one or more binding sites for the growing HA chain, and the ability to transfer this HA chain within the enzyme, to set up the next round of saccharide addition. The UDP-sugar substrates are produced and used by the HA synthase inside the cell, and the HA chain is continuously transferred across the membrane so that it is extruded to the cell exterior. In the case of bacteria, this HA forms a capsule. In the case of many eukaryotic cells, the result is either the formation of a pericellular HA coat surrounding the cell or the release and deposition of HA into the surrounding extracellular matrix (ECM).
Despite successful primary tumor treatment, the development of pulmonary
metastasis continues to be the most common cause of mortality in osteosarcoma
patients. A conventional drug development path ...requiring drugs to induce
regression of established lesions has not led to improvements for osteosarcoma
patients in over 30 years. Based on our growing understanding of metastasis
biology, it is now reasonable and essential that we focus on developing
therapeutics that target metastatic progression. To advance this agenda a
meeting of key opinion leaders and experts in the metastasis and osteosarcoma
communities was convened in Bethesda Maryland. The goal of this meeting was to
provide a “Perspective” that would establish a preclinical
translational path that could support the early evaluation of potential
therapeutic agents that uniquely target the metastatic phenotype. Although
focused on osteosarcoma the need for this perspective is shared among many
cancer types. The consensus achieved from the meeting included the following:
That the biology of metastatic progression is associated with
metastasis-specific targets/processes that may not influence grossly detectable
lesions; targeting of metastasis-specific processes is feasible; rigorous
preclinical data is needed to support translation of metastasis-specific agents
into human trials where regression of measurable disease is not an expected
outcome; preclinical data should include an understanding of mechanism of
action, validation of pharmacodynamic markers of effective exposure and
response, the use of several murine models of effectiveness, and where feasible
the inclusion of the dog with naturally occurring osteosarcoma to define the
activity of new drugs in the micro-metastatic disease setting.
Isolated rat hepatocytes bind to synthetic flat polyacrylamide matrices containing covalently attached galactose residues in a sugar-specific and concentration-dependent manner. Cell binding is ...mediated by the asialoglycoprotein receptor and occurs as a threshold response at or above a critical concentration of sugar in the matrix. Hepatocytes in the presence or absence of serum were able to spread on these synthetic galactose surfaces and were morphologically similar to cells on tissue culture plastic. Cell spreading also occurred as a threshold response but at a much higher critical concentration of sugar than for the cell-binding response. Above the critical concentration for spreading, the area occupied by a cell increased as the sugar concentration increased. By manipulating the galactose content of the matrix, cell spreading and cell binding can be differentiated as independent and separable threshold responses to the extracellular substratum.
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