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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.
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PMID:A comparison of binding properties and structure of NGF receptor on PC12 pheochromocytoma and A875 melanoma cells. 632 98

The PC12 clone of pheochromocytoma cells undergoes neuronal differentiation in the presence of nerve growth factor (NGF). Concomitant with this is a significant induction in the incorporation of radiolabeled fucose or glucosamine into a 230,000-dalton cell surface glycoprotein named the NGF-Inducible Large External, or NILE, glycoprotein (GP) (McGuire, J. C., L. A. Greene, and A. V. Furano (1978) Cell 15: 357-365). In the current studies NILE GP was purified from PC12 cells using wheat germ agglutinin-agarose affinity chromatography and SDS-polyacrylamide gel electrophoresis (PAGE). Polyclonal antisera were raised against purified NILE GP and were found to selectively immunoprecipitate a single 230,000-dalton protein from detergent extracts of PC12 cells metabolically labeled with either [3H]fucose, [3H]glucosamine, or [35S]methionine. These antisera stained the surfaces of PC12 cells by indirect immunofluorescence and were cytotoxic to PC12 cells in the presence of complement. Limited treatment of PC12 cells with either trypsin or pronase produced a fucosylated 90,000-dalton immunoreactive fragment of NILE GP which remained in the membrane. Using quantitative immunoelectrophoresis, the action of NGF on NILE GP was represent an increase in the amount of protein, rather than a selective increase in carbohydrate incorporation. Immunofluorescent staining of primary cell cultures and tissue whole mounts revealed that immunologically cross-reactive NILE GP appears to be expressed on the cell surfaces (somas and neurites) of most if not all peripheral and central neurons examined. Immunoprecipitation of radiolabeled cultures showed that the cross-reactive material had an apparent molecular weight by SDS-PAGE of 225,000 to 230,000 in the peripheral nervous system and 200,000 to 210,000 in the central nervous system. NILE-cross-reactive material was also found to a small extent on Schwann cell surfaces, but not at all on a variety of other cell types. These results suggest that immunoreactive NILE GP is distributed widely and selectively on neural cell surfaces.
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PMID:Nerve growth factor-inducible large external (NILE) glycoprotein: studies of a central and peripheral neuronal marker. 633 60

A serum-free culture of dissociated neurons from embryonic rat hippocampus has been established as a rapid and quantitative in vitro test system for neurotrophic signals in the mammalian brain. By means of this cell culture bioassay, a novel low molecular weight neurotrophic factor (NTF) could be identified. NTF is essential for in vitro brain neuron development, promoting survival and neurite outgrowth. The diffusible factor is synthesized and secreted into serum-free defined medium by cultured astrocytes from rat cerebral hemispheres. The number of viable neurons responding to NTF by neurite outgrowth is dependent on the concentration of the factor. Fractionation of astroglial conditioned medium by gel filtration on columns of Sephadex G-10 recovered biological activity of NTF in a single sharp peak corresponding to an apparent molecular weight of approximately equal to 500. NTF is stable to heat and cold and resistant to trypsin and pronase. Unlike nerve growth factor, NTF has no apparent effect on the neurite outgrowth of peripheral neurons. NTF-like activity is present in situ in the mammalian brain, in certain other nonneural tissues, and in C6 and B12 glioma cell conditioned media.
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PMID:Neurotrophic factor for central neurons. 636

In the pig, submandibular, native pancreatic, and urinary kallikreins are the same protein, consisting of a single polypeptide chain (alpha-kallikrein). Porcine tissue kallikrein shows very extensive sequence homology with several enzymes from submandibular glands of rats and mice, tonin, nerve growth factor gamma subunit, and submandibular proteinase A, nearly as high as with human urinary or rat submandibular kallikrein. Porcine pancreatic kallikrein isolated from partial autolyzates of pancreas carries an intrachain split (beta-kallikrein). Both chains exist in a high and low molecular weight form each because of differences in their carbohydrate content and form four types of pancreatic beta-kallikrein (B, A, III, and C). One cause of the narrow specificity of tissue kallikrein is their pronounced secondary specificity for a bulky, hydrophobic amino acid residue in P2. The hydrolysis of 10 peptide esters Ac-X-ArgOMe with different amino acids in P2 by porcine pancreatic kallikrein also showed distinct individual influences, the most favorably residues being phenylalanine and leucine as they occur in bovine kininogen. In contrast, specificity constants for hydrolysis by the digestive enzyme trypsin are similar for all these compounds. A peptide with the amino acid sequence around the methionyl bond cleaved in kininogen is also hydrolyzed by pancreatic kallikrein at this bond, but with a specificity constant three orders of magnitude lower. The lack of cleavage at lysine leading to the release of kallidin instead of bradykinin is due to the inability of porcine pancreatic kallikrein to accommodate an Arg-Pro leaving group.
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PMID:Enzymology of porcine tissue kallikrein. 655 41

In the presence of nerve growth factor (NGF), PC12 pheochromocytoma cells undergo neuronal differentiation with a concomitant 3- to 5-fold increase in the specific level of an Mr = 230,000 cell surface component named the NGF-inducible large external, or NILE, glycoprotein. Antisera raised against NILE glycoprotein (NILE GP) purified from PC12 cells have been found to recognize most, if not all, neurons derived from the peripheral and central nervous systems. In the current studies several of the biochemical properties of NILE GP were investigated. NILE GP was found to be phosphorylated in NGF-treated and -untreated PC12 cells and in cultured rat sympathetic neurons. The phosphate moiety of NILE GP is almost completely alkali labile, suggesting that phosphoserine groups predominate. Immunoprecipitation experiments revealed that incorporation of [32P]phosphate into NILE GP relative to total PC12 cell phosphoprotein was not significantly altered at 12 and 24 hr of NGF treatment but was enhanced 3-fold after 7 days and up to 5-fold after 2 to 3 weeks of NGF exposure. These changes in phosphorylated NILE GP paralleled, and therefore appeared to be mainly a consequence of, the NGF-induced increase in total cellular levels of NILE GP. By two-dimensional gel analysis, anti-NILE GP selectively immunoprecipitated two NGF-inducible spots (apparent Mr = 230,000; pI = 6.4 to 6.6) from PC12 cells labeled with either [3H] fucose, [35S]methionine, or [32P]phosphate. Anti-NILE GP immunoprecipitated a single band (apparent Mr = 205,000) from extracts of rat brain labeled with [3H] glucosamine. This confirms the previously established apparent molecular weight difference between central and peripheral NILE GP cross-reactive material. When PC12 cells, cerebellar cultures, and cultured cerebral cortex were treated with tunicamycin and labeled with [35S]methionine, nonglycosylated bands each with Mr = 160,000 were immunoprecipitated, implying that the differences in the mobilities on sodium dodecyl sulfate gels of cross-reactive NILE GP from different tissues is due to variation in glycosylation rather than to large differences in apoprotein structure. Prolonged treatment of PC12 cells with trypsin produced an immunoreactive fragment of NILE GP of apparent Mr = 28,000 that was phosphorylated but not glycosylated, and that remained in the membrane. NILE GP remained predominantly membrane associated under a variety of aqueous extraction conditions, suggesting that it is an integral membrane protein.
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PMID:Biochemical properties of the nerve growth factor-inducible large external (NILE) glycoprotein. 665 95

The composition and biosynthesis of glycoproteins, proteoglycans, and gangliosides have been studied in a clonal line of rat pheochromocytoma (PC12) cells. Glycoproteins account for approximately 78% of the glucosamine-labeled complex carbohydrates found in the culture medium, together with 17% chondroitin sulfate and 5% heparan sulfate. 10% of the glycoproteins but less than 1% of the proteoglycans are released by trypsin treatment of the cells, whose complex carbohydrates are composed of 93% glycoproteins, 1.3% chondroitin sulfate, 3.4% heparan sulfate, and 2.6% of mono- and disialogangliosides. Sequential lectin affinity chromatography and alkali treatment of glycopeptides prepared from the medium, trypsin-releasable, membrane, and cell-soluble glycoproteins demonstrated that in all of the subfractions large tri- and tetraantennary complex oligosaccharides account for 82 to 97% of those present in PC12 cell glycoproteins. Biantennary oligosaccharides account for approximately 2-6% of those in medium and trypsinate, as compared to 10-13% in the membrane and cell soluble glycoproteins, and there were large differences (ranging from 7 to 60%) in the proportions of biantennary oligosaccharides which are substituted by fucose on the core N-acetylglucosamine which is linked to asparagine. High mannose oligosaccharides are present predominantly in the cell membrane and soluble glycoproteins, where they account for 4 to 5% of the total glycoprotein labeling. In response to nerve growth factor (NGF), the PC12 cells extend long processes and acquire other properties similar to those of differentiated sympathetic neurons. Significant alterations were also observed in the complex carbohydrates of NGF-treated cells, the most striking of which were an almost 3-fold increase in labeled gangliosides and a 75% increase in trypsin-releasable glycoproteins. Cellular heparan sulfate decreased by 70% in response to NGF and increased by an equivalent amount in the culture medium, whereas an NGF-induced increase in chondroitin sulfate labeling occurred specifically in the cell membranes.
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PMID:Complex carbohydrates of cultured PC12 pheochromocytoma cells. Effects of nerve growth factor and comparison with neonatal and mature rat brain. 683 45

The pheochromocytoma PC12 cell possesses specific cell surface receptors that bind nerve growth factor (NGF) with two different affinities. The rate of dissociation of NGF from the higher affinity receptor is slower than from the lower affinity receptor; this rate is reduced to essentially zero at low temperature, allowing the extent of high-affinity binding to be determined. When NGF is added to PC12 cells, only low-affinity binding is observed. After a short lag period, high-affinity binding also appears and increases slowly. If NGF is removed from the medium after binding is initiated, high-affinity receptors continue to be formed at the expense of low-affinity receptors. The increase in receptor affinity is accompanied by a transfer of the NGF-receptor complex from a trypsin-sensitive to a trypsin-resistant state. This transfer does not involve internalization of the NGF. The data show that NGF binds first to receptors of low affinity and that the binding induces a conversion of a proportion of the receptors to a higher affinity state. It is also consistent with a model in which the change in affinity is due either to conformational changes in the receptor or to interaction of the occupied receptor with other receptors or with effector proteins in cell plasma membrane.
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PMID:Nerve growth factor receptors on PC12 cells: ligand-induced conversion from low- to high-affinity states. 693 21

The complete amino acid sequence of the gamma-subunit of mouse submaxillary gland 7 S nerve growth factor has been determined from analyses of the peptides generated by cyanogen bromide, trypsin, and chymotrypsin from the naturally occurring fragments. All peptides were sequenced automatically in a spinning-cup sequenator using Polybrene to minimize extractive losses by the solvents employed thoughout the degradation cycles. The gamma-subunit, a serine protease with arginine specificity, contains 233 amino acid residues and shares sequence homology with other proteases of this family. The five disulfide bonds of the gamma-subunit are a subset of the six disulfides present in bovine trypsin, as judged by the location of the half-cystine residues in the primary structure. An N-linked carbohydrate side chain is attached to Asn-78 in at least a majority of tee gamma-molecules.
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PMID:The amino acid sequence of the gamma-subunit of mouse submaxillary gland 7 S nerve growth factor. 726 6

Cultured corneal epithelial cells release a factor(s) that stimulates trigeminal neurons to form neurites in vitro. To characterize this trophic effect, conditioned media (serum free, supplemented) from cultures for corneal epithelium, stromal fibroblasts, and endothelium were studied further. Only epithelial conditioned medium (PCM) prolonged neuronal survival and induced neurite outgrowth. This trophic influence peaked after 2 to 3 days and gradually declined thereafter during a week when the medium was not renewed. Using a bioassay to score the percentage of initially viable neurons that extended neurites, it was found that the trophic effect of PCM was proportional to the conditioned medium concentration and to the cell density of the epithelial culture used for the conditioning. Maximum activity in PCM was correlated with confluency of the epithelial culture. Experiments using antiserum to nerve growth factor (NGF) and purified antibody to cold-insoluble globulin (CIG) indicated that the tropic effect of PCM was not derived from NGF or CIG. The trophic activity of PCM was abolished totally by heat or trypsin treatment but was not affected by collagenase. Although a fraction of the trophic activity was associated with the substratum after adsorption of PCM, this and other evidence did not suggest that the primary action of PCM was to enhance neuronal adhesion.
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PMID:Action of a trophic factor(s) from rabbit corneal epithelial culture on dissociated trigeminal neurons. 728 78

In murines, interleukins (IL) 3, 4, 9, and 10, nerve growth factor, and stem cell factor induce or promote growth and differentiation of mast cells (MC). Increased stimulation and synergy was observed when combinations of cytokines were used. In man, no growth factor for human MCs had been identified until recently, when SCF was found to induce in vitro growth and differentiation of human MCs. In the present study, the effects of recombinant human IL-3 and IL-4 on SCF-dependent differentiation of human MCs from their circulating progenitor cells in long-term culture were analyzed. Surprisingly, both IL-3 and IL-4 were found to inhibit SCF-dependent formation of human MCs (SCF, 100 ng/ml: 36.4 +/- 18.7 x 10(3)/ml; SCF + IL-3, 100 U/ml: 23.4 +/- 4.2 x 10(3)/ml; SCF + IL-4, 100 U/ml: 7.4 +/- 4.4 x 10(3)/ml) and synthesis of MC tryptase (SCF, 100 ng/ml: 73.2 +/- 17.6 ng/ml; SCF + IL-3, 100 U/ml: 10.8 +/- 3.1 ng/ml [p < 0.01]; SCF + IL-4, 100 U/ml: 8.1 +/- 1.5 ng/ml, [p = 0.02]). The inhibitory effects of these cytokines on SCF-dependent formation of human MCs were associated with an increase in the number of macrophages (IL-3) or lymphocytes (IL-4) in the same cultures and may be due to competitive recruitment of cells from a pool of multilineage hematopoietic progenitor cells.
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PMID:Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4. 752 90


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