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)

We have examined phosphorylation of nerve growth factor (NGF) receptor in cultured sympathetic neurons and PC12 cells. Dissociated rat superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and NGF receptor proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that NGF receptor components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or phorbol 12-myristate 13-acetate. When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only phosphoserine; the other peptide contained phosphoserine and phosphothreonine. No phosphotyrosine was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P]ATP and the cAMP-independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on serine and to a lesser extent on threonine. However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by cAMP-dependent protein kinase, casein kinase II, or protein kinase C (the Ca2+/phospholipid-dependent enzyme). We conclude that binding units of the NGF receptor are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.
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PMID:Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the cAMP-independent protein kinase FA/GSK-3. 302 30

The receptor for nerve growth factor (NGF) was purified from Triton X-100 extracts of sympathetic ganglia membranes by affinity chromatography on NGF-Sepharose. Elution of purified receptor was accomplished at pH 5 in the presence of 1 M NaCl. Sodium dodecyl sulfate gel electrophoresis of the purified iodinated receptor showed three major bands at Mr = 126,000, Mr = 105,000, and Mr = 81,000. Affinity labeling of the purified receptor using 125I-NGF and the photoreactive agent N-hydroxysuccinimidyl-p-azidobenzoate resulted in two major cross-linked complexes corresponding to Mr = 135,000 and Mr = 110,000. This labeling pattern is similar to that observed with sympathetic ganglia membranes (Massague, J., Guillette, B. J., Czech, M. P., Morgan, C. J., and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9419-9424) and indicates that these two forms do not arise from the cross-linking procedure. Reaction of the photoaffinity labeled NGF receptors with increasing amounts of trypsin resulted in a progressive decrease in the high molecular weight complex with a concomitant increase in the low molecular weight form. When the larger complex was isolated by electroelution from a sodium dodecyl sulfate gel and treated with trypsin, a species corresponding to Mr = 100,000 was generated. These observations are best explained by a precursor-product relationship for the two NGF receptor species of sympathetic neurons.
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PMID:Nerve growth factor receptor from rabbit sympathetic ganglia membranes. Relationship between subforms. 302 39

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.
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PMID:The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells. 303 14

Ciliary neurotrophic factor (CNTF) is a protein supporting the in vitro survival of a characteristic spectrum of embryonic chicken and rat peripheral neurons. High-speed supernatants of extracts from two neuroblastoma (NB) cell lines--the mouse C 1300 N2a and the human IMR 32--mimic the effects of CNTF on identical target neurons. Promotion of survival is dose-dependent with an ED50 of 80 micrograms (IMR 32) and 140 micrograms (C 1300 N2a) of protein per ml and saturable at plateau values for surviving neurons identical to those achieved with purified CNTF. Small amounts of a CNTF-like material are also detectable in medium conditioned by NB cells. The activity is destroyed by heat and trypsin and not blocked by antibodies to (mouse) nerve growth factor. Unlike the neurite-promoting and neuronal-survival modulating agent laminin, it cannot be depleted on poly(L-alpha-ornithine)-coated plastic surfaces. NB IMR 32 cell extracts were electrophoresed using NaDodSO4/PAGE and transferred to nitrocellulose. Ciliary ganglion neurons seeded on the blotting paper in culture medium lacking CNTF ("cell blot") exclusively survive on two distinct bands with apparent molecular masses of 24 and 48 kDa. Twenty-four kilodaltons is the molecular mass of a CNTF purified from rat sciatic nerve. These results suggest that NB cells may contain a CNTF-like protein and provide further evidence that neurons may store neurotrophic factors. Purified (chicken) CNTF failed to affect proliferation and neurite growth of NB cells. The biological relevance of CNTF for NB cells, therefore, remains to be elucidated.
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PMID:Neuroblastoma cells contain a trophic factor sharing biological and molecular properties with ciliary neurotrophic factor. 347 25

Cyanogen bromide cleavage followed by trypsin digestion of mouse nerve growth factor (NGF) allowed the isolation of a double chain unsymmetrical cystine peptide of high neurotrophic activity. The presence of tryptophan residues severely limits the synthetic approaches for selective disulfide bridging. The strategy applied for such a purpose as well as the preparation of the suitably protected tetradecapeptide corresponding to sequence 75-88 as key intermediate for the synthesis of the NGF fragment 10-25/75-88 are described.
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PMID:Synthesis of the trypsin fragment 10-25/75-88 of mouse nerve growth factor. I. The protected tetradecapeptide 75-88. 357 Jun 59

Normal human fibroblasts secrete a protein named protease nexin II (PN II) which previously was shown to form sodium dodecyl sulfate (SDS)-stable complexes with epidermal growth factor-binding protein (EGF-BP). These complexes then bind to the same cells and are rapidly internalized and degraded (Knauer, D.J., and Cunningham, D.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2310-2314). Here we describe a procedure for purifying PN II to apparent homogeneity from serum-free culture medium conditioned by human fibroblasts. The first step employed dextran sulfate-Sepharose affinity chromatography. Further purification was achieved by ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-400. Sequence analysis of purified PN II identified 33 amino-terminal amino acids; a computer search of several protein sequence data banks failed to reveal homologies with other reported amino acid sequences. Purified PN II had an apparent Mr of 106,000 and an isoelectric point of approximately 7.2. It retained full activity after incubation in the presence of 0.05% SDS or at a pH of 1.5. PN II formed SDS-stable complexes with EGF-BP, the gamma subunit of 7 S nerve growth factor, and trypsin with estimated Mr of 120,000, 120,000, and 110,000, respectively. PN II was metabolically labeled with [35S]methionine and purified; the metabolically labeled protein formed complexes with EGF-BP. Complexes between purified PN II and EGF-BP bound to human fibroblasts. These results show that the purified protein possesses the properties previously attributed to PN II in cell culture medium.
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PMID:Purification of protease nexin II from human fibroblasts. 359 85

Reverse-phase high-performance liquid chromatography was used to analyse the products of treatment of mouse nerve growth factor with cyanogen bromide followed by trypsin as described by Mercanti et al. All the biological activity was found to be due to incompletely cleaved starting material. Total digestion with trypsin led to complete loss of activity.
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PMID:On the non-existence of a tryptic peptide with biological activity derived from mouse nerve growth factor. 367 34

Neurite extension from sensory neuroblasts dissociated from chick embryo dorsal root ganglia can be stimulated by precoating the polylysine culture surface with extracts of skeletal muscle from bovine fetuses. The active factor(s) may be partially purified from cytosolic extracts of muscle by chromatography on Sepharose 6B and affinity chromatography on wheat germ agglutinin or Helix pomatia agglutinin columns. Extract concentrations of 10-50 micrograms protein per 1 ml were active in promoting neurite extension when the neurons were cultured without serum or nerve growth factor (beta NGF). However, levels of 1-10 micrograms/ml produced dramatic neurite extension when 10% (v/v) fetal or newborn calf serum or 0.5 ng/ml beta NGF was added to the medium. The biological activity was not blocked by antiserum that was raised against purified mouse laminin and that abolished the neurite-promoting activity of mouse laminin. The activity of the muscle extract was destroyed by trypsin or heparitinase, in contrast to the biological activity of purified mouse laminin, which was not abolished by heparitinase treatment. The activity could be resolved into two broad peaks on a Sepharose 2B column (apparent Mr between 2 X 10(6) and in 10 X 10(6) in native form). Treatment with dithiothreitol was necessary to dissociate the factor for electrophoresis in 4.25% polyacrylamide-SDS gels, revealing three major polypeptide bands at Mr = 160,000, 195,000 and 200,000. This preliminary characterization indicates that the neurite-promoting activity from bovine skeletal muscle tissue consists of a high-molecular-weight complex, one essential component of which is a heparan sulfate.
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PMID:Neurite-promoting activity from fetal skeletal muscle: partial purification of a high-molecular-weight form. 370 85

To analyze the interdependence of neurons and astroglia during central nervous system development, a rapid method for purifying early postnatal cerebellar neurons and astroglia, and recombining them in vitro, has been developed. The influence of neurons on astroglial shape and proliferation has been evaluated with an in vitro model system previously used to describe the role of cerebellar astroglia in neuronal migration and positioning (Hatten, M. E., and R. K. H. Liem, 1981, J. Cell Biol., 90:622-630; and Hatten, M. E., R. K. H. Liem, and C. A. Mason, 1984, J. Cell Biol., 98:193-204. Cerebellar tissue harvested from C57Bl/6J mouse cerebellum on the third or fourth day postnatal was dissociated into a single cell suspension with trypsin, and enriched glial and neuronal fractions were separated with a step gradient of Percoll. Highly purified astroglial and neuronal fractions resulted from subsequently preplanting the cells on a polylysine-coated culture surface. In the absence of neurons, astroglia, identified by staining with antisera raised against purified glial filament protein, assumed a flattened shape and proliferated rapidly. In the absence of astroglia, cerebellar neurons, identified by staining with antisera raised against the nerve growth factor-inducible large external (NILE) glycoprotein and by electron microscopy, formed cellular reaggregates, had markedly impaired neurite outgrowth, and survived poorly. When purified neurons and isolated astroglia were recombined, astroglial proliferation slowed markedly and the flattened shape expressed in the absence of neurons transformed into highly elongated profiles that resembled embryonic forms of cerebellar astroglia. After longer periods (48-72 h) in the presence of neurons, astroglia had "Bergmann-like" or "astrocyte-like" shapes and neurons commonly associated with them. These results suggest that neurons influence the differentiation of astroglia.
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PMID:Neuronal regulation of astroglial morphology and proliferation in vitro. 388 55

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.
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PMID:Protease nexin. Properties and a modified purification procedure. 399 57


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