Identification of carbohydrate binding protein 35 in heterogeneous nuclear ribonucleoprotein complex

In previous studies, a lectin designated as carbohydrate binding protein 35 (CBP35) was identified in the nucleus and cytoplasm of cultured mouse 3T3 fibroblasts. In the present study, we observed that treatment of Triton X-100 permeabilized 3T3 cells with ribonuclease A released CBP35 from the nuclei, while parallel treatment with deoxyribonuclease I failed to do so. This conclusion was based on (a) immunofluorescence analysis of the nuclear residue after detergent and enzymatic treatments and (b) immunoblotting analysis of the supernatant fraction produced by these treatments. These results indicate that CBP35 may be associated with the ribonucleoprotein elements of the 3T3 cell nuclei. In corroboration with this conclusion, fractionation of the nucleoplasm derived from 3T3 cells on a cesium sulfate gradient (1.25-1.75 g/mL) localized CBP35 in fractions with densities of 1.30-1.32 g/mL, corresponding to the range of densities reported for heterogeneous nuclear ribonucleoprotein complex (hnRNP). Conversely, when nucleoplasm was fractionated on an affinity column of Sepharose derivatized with N-(epsilon-aminocaproyl)-D-galactosamine, the bound and eluted fraction contained RNA, as well as a set of polypeptides whose molecular weights matched those reported for the core particle of hnRNP. One of these polypeptides was identified as CBP35. These results suggest that CBP35 is a component of hnRNP.     

Carbohydrate-binding protein 35: identification of the galactose-specific lectin in various tissues of mice.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured mouse 3T3 fibroblasts. In this study, antibodies directed against CBP35 were used to screen for cross-reactive proteins in various cultured cells and in various organs and tissues of mice. Cross-reactive proteins of the same molecular weight (Mr, 35,000) were found in human, mouse, and chicken fibroblasts and in a macrophage-like cell line, P388D1. Similarly, cross-reactive proteins were also found in the embryonic liver, lung, spleen, thymus, skin, and muscle tissue and in the lung, artery, thymus, and spleen of the adult mouse. Fractionation of extracts of mouse lung on affinity columns of asialofetuin-Sepharose yielded a protein whose molecular weight, carbohydrate-binding specificity, and immunological properties suggest that it is CBP35 derived from the lung, hereafter designated CBP35 (lung). The binding of 125I-labeled CBP35 (lung) to rabbit erythrocytes was quantitated in the presence and absence of various carbohydrates. It was found that only carbohydrates containing galactose were inhibitors of the binding; the disaccharide lactose was 100-fold more potent as an inhibitor than was the monosaccharide galactose. When extracts of the adult mouse liver were fractionated by asialofetuin-Sepharose chromatography, only a protein corresponding to CBP16 was isolated; no CBP35 was found. These results corroborate the immunoblotting data, which indicated that CBP35 was not detectable in the adult mouse liver.     

Intrinsic Signals in the Unique Domain Target p56lckto the Plasma Membrane Independently of CD4

In T lymphocytes, the Src-family protein tyrosine kinase p56^lck (Lck) is mostly associated with the cytoplasmic face of the plasma membrane. To determine how this distribution is achieved, we analyzed the location of Lck in lymphoid and in transfected nonlymphoid cells by immunofluorescence. We found that in T cells Lck was targeted correctly, independently of the cell surface proteins CD4 and CD8 with which it interacts. Similarly, in transfected NIH-3T3 fibroblasts, Lck was localized at the plasma membrane, indicating that T cell–specific proteins are not required for targeting. Some variation in subcellular distribution was observed when Lck was expressed in HeLa and MDCK cells. In these cells, Lck associated with both the plasma membrane and the Golgi apparatus, while subsequent expression of CD4 resulted in the loss of Golgi-associated staining. Together, these data indicate that Lck contains intrinsic signals for targeting to the plasma membrane. Furthermore, delivery to this site may be achieved via association with exocytic transport vesicles.

A mutant Lck molecule in which the palmitoylation site at cysteine 5 was changed to lysine (LC2) localized to the plasma membrane and the Golgi region in NIH3T3 cells. However, the localization of a mutant in which the palmitoylation site at cysteine 3 was changed to serine (LC1) was indistinguishable from wild-type Lck. Chimeras composed of only the unique domain of Lck linked to either c-Src or the green fluorescent protein similarly localized to the plasma membrane of NIH-3T3 cells. Thus, the targeting of Lck appears to be determined primarily by its unique domain and may be influenced by the use of different palmitoylation sites.

     

Syntaxin 4 in 3T3-L1 adipocytes: regulation by insulin and participation in insulin-dependent glucose transport.

Syntaxins are thought to be membrane receptors that bind proteins of the synaptobrevin/vesicle-associated membrane protein (VAMP) family found on transport vesicles. Recently, we detected synaptobrevin II and cellubrevin on immunopurified vesicles containing the glucose transporter 4 (GLUT4) in insulin-responsive cells. In an effort to identify the plasma membrane receptors for these vesicles, we now examine the expression of syntaxins in the 3T3-L1 adipocyte cell line. Neither syntaxin 1A nor 1B was found, in keeping with the neuronal restriction of these isoforms. In contrast, syntaxins 2 and 4 were readily detectable. By subcellular fractionation and estimation of protein yields, 67% of syntaxin 4 was localized to the plasma membrane, 24% to the low-density microsomes, and 9% to the high-density microsomes. Interestingly, acute insulin treatment decreased the content of syntaxin 4 in low-density microsomes and caused a corresponding gain in the plasma membrane fraction, reminiscent of the recruitment of GLUT4 glucose transporters. In contrast, there was no change in the distribution of syntaxin 2, which was mostly associated in the plasma membrane. A fraction of the intracellular syntaxin 4 was recovered with immunopurified GLUT4-containing vesicles. Moreover, anti-syntaxin 4 antibodies introduced in permeabilized 3T3-L1 adipocytes significantly reduced the insulin-dependent stimulation of glucose transport, in contrast to the introduction of irrelevant immunoglobulin G, which was without consequence. We propose that either the plasma membrane and/or the vesicular syntaxin 4 are involved in docking and/or fusion of GLUT4 vesicles at the cell surface of 3T3-L1 adipocytes.     

Immunocytochemical identification of the human α2-macrogiobulin receptor in monocytes and fibroblasts: Monoclonal antibodies define the receptor as a monocyte differentiation antigen

The α₂-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-α₂-macroglobulin receptor monoclonal antibodies bind to 90–100% of the blood monocyte population and not to other blood cells. This defines the α₂-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18–33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Immunofluorescence localization of phosphotyrosine containing proteins in RSV-transformed mouse fibroblasts

The localization of phosphotyrosine-containing proteins (P-TYR proteins) was studied by immunofluorescence microscopy employing affinity-purified azobenzyl phosphonate (ABP) antibodies, which specifically cross-react with phosphotyrosine. In Rous sarcoma virus (RSV)-transformed fibroblasts, after fixation followed by permeabilization with nonionic detergents, ABP antibodies gave a diffuse staining of the cytoplasm and specifically decorated restricted areas of the ventral plasma membrane corresponding to adhesion plaques, identified by interference reflection microscopy and staining with anti-vinculin serum. Specific decoration was also observed at the level of cell-cell contacts and at the tips of filopodial protrusions. Control non-transformed 3T3 mouse fibroblasts were not significantly stained by ABP antibodies. These findings show that, in RSV-transformed cells, proteins phosphorylated at tyrosine residues are found at cell-substratum and cell-cell contacts.     

Intranuclear Distribution of Galectin-3 in Mouse 3T3 Fibroblasts: Comparative Analyses by Immunofluorescence and Immunoelectron Microscopy

The intracellular distribution of carbohydrate binding protein 35 (CBP35), recently named galectin-3, was studied in mouse 3T3 fibroblasts, using immunofluorescence at the light microscope level and immunogold labeling at the ultrastructural level. In general, serum-stimulated, proliferating cells showed higher levels of labeling than quiescent cultures of the same cells. In the proliferating cells, the labeling intensity was higher in the nucleus than in the cytoplasm. Treatment of permeabilized cells or thin sections with ribonuclease A decreased the immunolabeling intensity, whereas parallel control treatments with deoxyribonuclease I failed to yield the same effect. While there appears to be general agreement between the immunofluorescence and the ultrastructural results regarding the level of CBP35 and its association with nuclear ribonucleoprotein complexes, there was one striking difference in terms of labeling of specific subnuclear structures. Immunofluorescence results indicate diffuse distribution of CBP35 within the nucleus, but the label appears to be excluded from certain "black holes," which most probably correspond to nucleoli. On the other hand, immunogold particles were observed in electron microscopy, mainly in interchromatin spaces, except for interchromatin granule clusters, at the border of condensed chromatin, on the dense fibrillar component, and at the periphery of the fibrillar centers of nucleoli.     

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins. J. Cell. Biochem. 68:139–150, 1998. © 1998 Wiley-Liss, Inc.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.     

Carbohydrate-binding protein 35. Isoelectric points of the polypeptide and a phosphorylated derivative

Purified carbohydrate-binding protein 35 (CBP35) and extracts of mouse cells containing CBP35 were analyzed by two-dimensional gel electrophoresis. Such an analysis on recombinant CBP35, obtained by expression of a cDNA clone in Escherichia coli, yielded a pI value of 8.7. When extracts of mouse 3T3 cells were subjected to two-dimensional gel electrophoresis and immunoblotting, two spots were observed, corresponding to pI values of 8.7 and 8.2. The pI 8.2 species represents post-translational modification of the CBP35 polypeptide (pI 8.7) by the addition of a single phosphate group. This conclusion is based on the finding that purified CBP35 contained a pI 8.2 species that was labeled with 32PO4 and could be converted to the unlabeled pI 8.7 species by alkaline phosphatase treatment. The phosphorylated (pI 8.2) form of CBP35 is found in both the cytosolic and nuclear fractions, whereas the unphosphorylated (pI 8.7) species is found exclusively in the nuclei. Quiescent populations of 3T3 fibroblasts (confluent monolayers or serum-starved sparse cultures) are characterized by the predominance of phosphorylated CBP35. Stimulation of the same cells into the proliferative state resulted in an increase in the amount of the phosphorylated species; more dramatic, however, is the elevation of the level of the unphosphorylated form, which is barely detectable in quiescent cells.     

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Preferential degradation of the terminal carbohydrate moiety of membrane glycoproteins in rat hepatoma cells and after transfer to the membranes of mouse fibroblasts

Glycoproteins in the plasma membrane of rat hepatoma cells were labeled at their externally exposed tyrosine residues with 131I and at their galactose and sialic acid residues with 3H. The degradation of both isotopes in the total cell protein fraction, in glycoproteins purified by concanavalin A, and in glycoproteins separated on two-dimensional gels was determined. Similarly, the total cellular membrane glycoproteins were metabolically labeled with [35S]methionine and [3H]fucose. The fate of both incorporated labels was followed by lectin chromatography or by precipitation of the proteins with specific antibodies followed by electrophoretic gel separation. In both labeling experiments, the carbohydrate markers were lost from the ligand- recognized fraction with similar kinetics as from the total cell protein fraction. In some glycoprotein species which were separated by two-dimensional gel electrophoresis, the polypeptide portion exhibited up to a twofold slower rate of degradation relative to that of the carbohydrate moiety. This difference is most pronounced in carbohydrate- rich glycoproteins. To corroborate this finding, double-labeled membrane glycoproteins were incorporated into reconstituted phospholipid vesicles which were then transferred via fusion into the plasma membrane of mouse fibroblasts. Both the polypeptide and carbohydrate moieties of the transferred membrane glycoproteins were degraded with the same relative kinetics as in the original hepatoma cells. The rate of degradation is mostly a function of the structural properties of the membrane components as shown by the preservation of metabolically stable fucogangliosides of Reuber H-35 hepatoma cells transferred onto the fibroblasts. The technique of insertion of membrane components into the plasma membrane of another cell should assist in the elucidation of the exact route and mechanism of membrane protein destruction.     

Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.     

Immunocytochemical identification of the human alpha 2-macroglobulin receptor in monocytes and fibroblasts: monoclonal antibodies define the receptor as a monocyte differentiation antigen

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Glycosylated nuclear lectin CBP70 also associated with endoplasmic reticulum and the Golgi apparatus: does the "classic pathway" of glycosylation also apply to nuclear glycoproteins?

The subcellular plurilocalization of some lectins (galectin-1, galectin-3, galectin-10, calreticulin, etc.) is an intriguing problem, implying different partners according to their localization, and involvement in a variety of cellular activities. For example, the well-known lectin, galectin-3, a lactose-binding protein, can act inside the nucleus in splicing events, and at the plasma membrane in adhesion, and it was demonstrated that galectin-3 interacts in the cytoplasm with Bcl-2, an antiapoptotic protein. Some years ago, our group isolated a nuclear lectin CBP70, capable of recognizing N-acetylglucosamine residues. This lectin, first isolated from the nucleus of HL60 cells, was also localized in the cytoplasm. It has been demonstrated that CBP70 is a glycosylated lectin, with different types of glycosylation, comparing cytoplasmic and nuclear forms. In this article, we have studied the localization of CBP70 in undifferentiated HL60 cells by electron microscopy, immunofluorescence analysis, and subcellular fractionation. The results obtained clearly demonstrated that CBP70 is a plurilocalized lectin that is found in the nucleus, at the endoplasmic reticulum, the Golgi apparatus, and mitochondria, but not at the plasma membrane. Because CBP70, a nuclear glycoprotein, was found to be associated also with the endoplasmic reticulum and the Golgi apparatus where the glycosylation take place, it raised the question: where does the glycosylation of nuclear proteins occur?     

Subcellular localisation of the middle and large T-antigens of polyoma virus.

The distribution of two of the polyoma virus early proteins (the large and middle T-antigens) in lytically infected mouse cells and transformed rat cells has been investigated by indirect immunofluorescence and immuno-electron microscopy using well-characterised monoclonal antibodies. By these techniques, the viral large T-antigen was found almost exclusively in the nucleus, sometimes in association with nuclear pores, but never in the nucleolus. In lytically infected, but not transformed cells, fluorescence was detected in discrete areas ('hot spots') within the nucleus and, in a minor population of lytically infected cells, cytoplasmic immunoreactive material was observed. The viral middle T-antigen was found in association with most cytoplasmic membranes and in the majority of cells mainly in the endoplasmic reticulum. Only a fraction of the staining was observed in the plasma membrane and no staining in the nucleoplasm was observed. The data suggest that the site of action of the major transforming activity of polyoma virus need not be at the plasma membrane. Functions associated with the viral antigens are discussed in terms of their subcellular distributions within cells.     

Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts

Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF-stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane.     

Calcium-binding proteins 33 kDa, 35 kDa, and 65/67 kDa in normal rat and Morris hepatoma tissues. A biochemical and immunohistochemical study

Polyclonal antibodies were raised against membrane-associated calcium-binding proteins (apparent molecular masses 65000 and 67000 (CBP 65/67) and 33000 and 35000 (CBP 33 and CBP 35)), which were isolated from rat liver and Morris hepatoma. Using immunoblotting, various amounts of CBP 33 and CBP 35 as well as CBP 65/67 were detected in most rat organs. Using alkaline phosphatase and monoclonal-anti-alkaline phosphatase antibodies (APAAP), all the calcium-binding proteins were detected by immunohistochemical techniques in the plasma membranes of many cells, such as vascular endothelial cells, lymphocytes, epididymal principal cells, secretory and excretory duct cells of certain exocrine glands, straight distal tubular cells of the kidney, and in the cytoplasm of muscle cells and fibres as well as nerve cells and chondrocytes, and in connective tissue elements. Immunohistochemical analysis also showed that in polarized epithelial cells, e.g., renal tubular cells, epididymal principal cells or excretory duct cells, these calcium-binding proteins are present exclusively or mostly in the luminal plasma membrane.