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)

The established process for iron uptake into mammalian cells involves transferrin and its receptor. Here, the role of the glycosyl-phosphatidylinositol (GPI)-linked transferrin homologue, melanotransferrin or p97, was studied using CHO cell lines defective in the transferrin receptor (TR) and transfected with human TR and/or human p97. The presence of p97 doubled the iron uptake, which could be explained by the binding of one atom of iron to one molecule of p97. The internalization of iron was shown to be temperature sensitive and saturated at a media iron concentration of 2.5 micrograms/ml with a Vmax of 0.1 pmol Fe/10(6) cell/min and a Km of 2.58 microM for p97. Treatment of the cells with either phosphatidylinositol-phospholipase C or monoclonal antibodies against p97 resulted in over a 50% reduction and a 47% increase in the iron uptake respectively. These data identify p97 as a unique cell surface GPI-anchored, iron binding protein involved in the transferrin-independent uptake of iron in mammals.
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PMID:A novel iron uptake mechanism mediated by GPI-anchored human p97. 755 58

Melanotransferrin, also called p97, is a cell surface glycoprotein which was first described as a marker antigen for human melanoma cells. Although p97 has a striking structural similarity to human serum transferrin and lactoferrin, its function has not yet been determined. One feature that distinguishes p97 from the other members of the transferrin family is the presence of a stretch of 24 hydrophobic amino acids at the C terminus, previously assumed to form a proteinacious transmembrane domain. In this study, sensitivity to bacterial phosphatidylinositol-specific phospholipase C, biosynthetic labeling with [3H]ethanolamine, and partitioning in Triton X-114 are used to establish that p97 is expressed at the cell surface as a glycosylphosphatidylinositol-anchored protein. In addition, to gain insight into the intracellular transport of p97, biosynthetic transport studies were performed on a melanoma cell line. These studies resulted in the identification of an additional form of p97 which is found in the medium and which likely does not originate from an alternatively spliced form of the p97 mRNA. These findings, together with our recent observation of the co-localization of p97 and the transferrin receptor in brain capillary endothelium (W. A. Jefferies, M. R. Food, R. Gabathuler, S. Rothenberger, T. Yamada, and P. L. McGeer, manuscript submitted) raise important questions about the function of the two forms of p97 detected and the possible involvement of this protein in a cellular iron uptake mechanism that is independent from the transferrin/transferrin receptor system.
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PMID:Transport and expression in human melanomas of a transferrin-like glycosylphosphatidylinositol-anchored protein. 830 Jun 36

Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.
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PMID:Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells. 831

Stimulation of phospholipase C-gamma (PLC-gamma) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-gamma via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-gamma, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-gamma-associated proteins, RASM cells were labeled with [32P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-gamma was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-gamma-associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src-transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-gamma-associated protein that may be required for Ang II-mediated signal transduction.
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PMID:Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma-associated proteins. Characterization of a c-Src-dependent 97-kD protein in vascular smooth muscle cells. 931 36

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins.
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PMID:Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells. 1861 52

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 microg/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 microg/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum.
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PMID:Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers. 1861 45

Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL.
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PMID:Increasing GPI-anchored protein harvest concentrations from suspension and porous microcarrier CHO cell cultures. 1863 51