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61.
We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups. 相似文献
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Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells. 相似文献
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Golgi apparatus, isolated from rat liver, incorporate [14C]sialic acid from CMP[14C]sialic acid into endogenous glycolipid and glycoprotein acceptors. Incorporation of [14C]sialic acid into endogenous glycoprotein acceptors was stimulated an average of 3-fold by Triton X-100 at an optimal concentration of 0.05% and was inhibited at higher concentrations. Incorporation of [14C]sialic acid into endogenous glycolipid acceptors was not stimulated by detergent. The major glycolipid product was identified by thin-layer chromatography as the ganglioside Gd3. SDS-polyacrylamide gel electrophoresis of the glycoprotein products demonstrated incorporation of [14C]sialic acid into 6–7 major bands. Neuraminidase studies determined that approximately 60% of the [14C]sialic acid incorporated into endogenous acceptors in the absence of detergent had a luminal orientation. Furthermore, electron microscopy studies showed that the isolated Golgi apparatus fraction consisted of intact membrane cisternae. Our results demonstrate that sialylation of cisternal acceptors located on the inside of the membrane occurs in the absence of detergent. They are consistent with carrier-mediated transport as a mechanism to allow CMPsialic acid to traverse the Golgi apparatus membrane and to be used to glycosylate endogenous glycoprotein and glycolipid acceptors. 相似文献
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Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy 总被引:3,自引:2,他引:1
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Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined. 相似文献
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