Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treating the liposome-intercalatable heparan sulfate proteoglycans from human lung fibroblasts and mammary epithelial cells with heparitinase and chondroitinase ABC revealed different core protein patterns in the two cell types. Lung fibroblasts expressed heparan sulfate proteoglycans with core proteins of approximately 35, 48/90 (fibroglycan), 64 (glypican), and 125 kDa and traces of a hybrid proteoglycan which carried both heparan sulfate and chondroitin sulfate chains. The mammary epithelial cells, in contrast, expressed large amounts of a hybrid proteoglycan and heparan sulfate proteoglycans with core proteins of approximately 35 and 64 kDa, but the fibroglycan and 125-kDa cores were not detectable in these cells. Phosphatidylinositol-specific phospholipase C and monoclonal antibody (mAb) S1 identified the 64-kDa core proteins as glypican, whereas mAb 2E9, which also reacted with proteoglycan from mouse mammary epithelial cells, tentatively identified the hybrid proteoglycans as syndecan. The expression of syndecan in lung fibroblasts was confirmed by amplifying syndecan cDNA sequences from fibroblastic mRNA extracts and demonstrating the cross-reactivity of the encoded recombinant core protein with mAb 2E9. Northern blots failed to detect a message for fibroglycan in the mammary epithelial cells and in several other epithelial cell lines tested, while confirming the expression of both glypican and syndecan in these cells. Confluent fibroblasts expressed higher levels of syndecan mRNA than exponentially growing fibroblasts, but these levels remained lower than observed in epithelial cells. These data formally identify one of the cell surface proteoglycans of human lung fibroblasts as syndecan and indicate that the expression of the cell surface proteoglycans varies in different cell types and under different culture conditions.
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PMID:Differential expression of cell surface heparan sulfate proteoglycans in human mammary epithelial cells and lung fibroblasts. 133 31

The glycosylphosphatidylinositol (GPI)-anchor of the plasma membrane-associated heparan sulfate (HS) proteoglycan was metabolically radiolabeled with [3H]myristic acid, [3H]palmitic acid, [3H]inositol, [3H]ethanolamine, or [32P]phosphate in rat ovarian granulosa cell culture. Cell cultures labeled with [3H]myristic acid or [3H]palmitic acid were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100 and the proteoglycans were purified by ion exchange chromatography after extensive delipidation. Specific incorporation of 3H into GPI-anchor was demonstrated by removing the label with a phosphatidylinositol-specific phospholipase C (PI-PLC). Incorporation of 3H activity into glycosaminoglycans and core glycoproteins was also demonstrated. However, the specific activity of 3H in these structures was approximately 2 orders of magnitude lower than that in the GPI-anchor, suggesting that 3H label was the result of the metabolic utilization of catabolic products of the 3H-labeled fatty acids. PI-PLC treatment of cell cultures metabolically labeled with [3H]inositol, [3H]ethanolamine, or [32P]phosphate specifically released radiolabeled cell surface-associated HS proteoglycans indicating the presence of GPI-anchor in these proteoglycans. GPI-anchored HS proteoglycans accounted for 20-30% of the total cell surface-associated HS proteoglycans and virtually all of them were removed by PI-PLC. These results further substantiate the presence of GPI-anchored heparan sulfate proteoglycan in ovarian granulosa cells and its cell surface localization.
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PMID:Metabolic labeling of glycosylphosphatidylinositol-anchor of heparan sulfate proteoglycans in rat ovarian granulosa cells. 153 35

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.
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PMID:Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. 153 36

The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line [UMR 106-01 (BSP)] were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied. HS proteoglycans were metabolically labelled by incubation of cell cultures with [3H]glucosamine or [3H]leucine and [35S]sulphate. HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa). The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable. Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-[125I]iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains. Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the [125I]TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane. Approx. 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins. In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased. These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain. These observations are similar to those reported for ovarian granulosa cells [Yanagishita & McQuillan (1989) J. Biol. Chem. 264 17551-17558], and thus may represent a general phenomenon for many cell types.
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PMID:Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP). 163 8

We report the identification of cell surface glycoproteins that bind transforming growth factor-beta (TGF-beta) in an isoform-specific manner, and are distinct from TGF-beta receptors I and II or the TGF-beta binding proteoglycan beta-glycan. The novel TGF-beta binding proteins have been identified in various cell lines including fetal bovine heart endothelial cells and MG-63 human osteosarcoma cells. They include proteins of 90-100 and 180 kDa that preferentially bind TGF-beta 1 (KD 0.1-0.2 nM) and proteins of 60 and 140 kDa that preferentially bind TGF-beta 2 (KD 0.5-1 nM). The 180-kDa TGF-beta 1 binding protein and the 60- and 140-kDa TGF-beta 2 binding proteins can be released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, suggesting that these proteins are attached to the plasma membrane through a phosphatidylinositol anchor. The expression of these three proteins as well as their sensitivity to phosphatidylinositol-specific phospholipase C is cell line-dependent. The 90-100-kDa TGF-beta 1 binding proteins are components of a 190-kDa disulfide-linked complex. The structural properties of these proteins and their high affinity and selectivity for different TGF-beta isoforms defines them as a novel class of cell surface TGF-beta binding proteins.
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PMID:Isoform-specific transforming growth factor-beta binding proteins with membrane attachments sensitive to phosphatidylinositol-specific phospholipase C. 165 36

Two mAbs raised against the 64-kD core protein of a membrane heparan sulfate proteoglycan from human lung fibroblasts also recognize a nonhydrophobic proteoglycan which accumulates in the culture medium of the cells. Pulse-chase studies suggest that the hydrophobic cell-associated forms act as precursors for the nonhydrophobic medium-released species. The core proteins of the medium-released proteoglycans are slightly smaller than those of the hydrophobic cell-associated species, but the NH2-terminal amino acid sequences of both forms are identical. The characterization of human lung fibroblast cDNAs that encode the message for these core proteins and the effect of bacterial phosphatidylinositol-specific phospholipase C suggest that the hydrophobic proteoglycan is membrane-anchored through a phospholipid tail. These data identify a novel membrane proteoglycan in human lung fibroblasts and imply that the shedding of this proteoglycan may be related to the presence of the phospholipid anchor.
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PMID:Molecular cloning of a phosphatidylinositol-anchored membrane heparan sulfate proteoglycan from human lung fibroblasts. 214 68

The effect of phosphatidylinositol-specific phospholipase C (PI-PLC) on the release of lipoprotein lipase was studied in F1 heart cell cultures. Exposure of the cultures for 10 min to PI-PLC resulted in a 2-fold increase in the release of lipoprotein lipase (LPL) into the culture medium. PI-PLC released LPL from the heparin-releasable pool and PI-PLC was not effective in cultures pretreated with heparin. Insulin had no influence on the release of LPL from the heart cell cultures, even though it enhanced the uptake of 2-deoxy[3H]glucose by these cells. In cultures labeled with 35S, treatment with PI-PLC resulted in an increase in the release of 35S-labeled proteoglycan. PI-PLC was also effective in enhancing the release of bovine LPL exogenously bound to cultured aortic smooth muscle cells. The findings that PI-PLC was not effective after heparin, that it did release exogenously added LPL to cell cultures and that it released 35S-labeled proteoglycan, were interpreted to indicate that PI-PLC apparently acts on the release of LPL in an indirect manner, releasing heparan sulphate to which LPL is bound. As there is a previously described correlation between circulating LPL and the heparin-releasable LPL, we hypothesize that the activity of PI-PLC in the endothelial cell membrane or plasma phosphatidyl-specific phospholipase D regulates the plasma LPL levels.
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PMID:Phosphatidylinositol-specific phospholipase C releases lipoprotein lipase from the heparin releasable pool in rat heart cell cultures. 255 75

A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with 35SO2/4-. The cells were treated with a number of ligands to displace the heparan 35SO4 proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO4 (50 mM), D-glucose-6-PO4 (50 mM), myo-inositol-2-PO4 (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO4 (1-2 mM). D-myo-Inositol-1,3,4-(PO4)3 (1 mM) and L-myo-inositol-1-PO4 (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomannan (4 mg/ml), D-xylose-1-PO4 (50 mM), D-glucose-6-SO4 (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When 35SO2/4- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [35SO4]HSPG that was released by myo-inositol-PO4 increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO4 was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO4, the release of the cell surface [35SO4]HSPG was increased to 73%. When the [35SO4]HSPG that was released from the cell surface by treatment with myo-inositol-PO4 was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO4 chains which appeared in the nucleus. Uptake was Ca2+- and Mg2+-independent. The amount of [35SO4]HSPG taken up was markedly reduced when the myo-inositol-PO4-releasable [35SO4]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO4, the amount of heparan SO4 found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO4 were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO4.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth. 295 71

The morphological changes that occur during intestinal development have been extensively described, but the molecular basis of these changes is largely unknown. As a result of our efforts to identify molecules that play a role in intestinal morphogenesis during development, we have previously isolated a cDNA that is developmentally regulated in the intestine. This cDNA, named OCI-5, was recently shown to have 20-25% identity at the protein-sequence level with glypican and cerebroglycan, two heparan sulphate proteoglycans (HSPG) that are attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Here we provide experimental evidence indicating that OCI-5 is also a GPI-linked HSPG. We demonstrate this by showing that OCI-5 can be labelled with radioactive sulphate and can be digested by heparitinase, but not by chondroitinase. We also show that treatment with phosphatidylinositol-specific phospholipase C releases OCI-5 from the cell surface of COS cells transfected with an OCI-5 expression vector. The identification of OCI-5 as a GPI-linked HSPG confirms that this proteoglycan belongs to the same family of HSPGs that include glypican and cerebroglycan.
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PMID:Identification of a new membrane-bound heparan sulphate proteoglycan. 748 96

We have used suramin and brefeldin A to investigate the nature of a heparan sulphate proteoglycan that appears to recycle from the cell surface to intracellular compartments which synthesize new heparan sulphate chains. Suramin, which would block internalization and deglycanation of a putative recycling cell surface proteoglycan, markedly increases the yield of a membrane-bound proteoglycan with a core protein of 60-70 kDa and unusually long heparan sulphate side chains. When transport of newly made core proteins to their Golgi sites for glycosaminoglycan assembly is blocked, by using brefeldin A, [3H]glucosamine and [35S]sulphate incorporation into cell surface-bound heparan sulphate proteoglycan can still take place. After chemical biotinylation of cell surface proteins in brefeldin A-treated cells, followed by metabolic [35S]sulphation in the presence of the same drug, biotin-tagged [35S]proteoglycan can be demonstrated, indicating the presence of recycling proteoglycan species. By pre-labelling cells with [3H]leucine or [3H]inositol in the presence of suramin, followed by chase labelling with [35S]sulphate in the presence of brefeldin A, a 3H- and 35S-labelled, hydrophobic heparan sulphate proteoglycan with a core protein of 60-65 kDa is obtained. The proteoglycan loses its hydrophobicity when glucosamine-inositol bonds are cleaved, indicating that it is membrane bound via a glycosylphosphatidylinositol anchor. However, treatment with phosphatidylinositol-specific phospholipase C has no effect, suggesting that the inositol moiety may be acylated. We propose that a portion of the lipid-anchored proteoglycan glypican is internalized, recycled via the Golgi, where heparan sulphate chains are added, and finally re-deposited at the cell surface.
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PMID:Recycling of a glycosylphosphatidylinositol-anchored heparan sulphate proteoglycan (glypican) in skin fibroblasts. 757 95


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