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)

Given the importance of the swine (Sus scrofa) as an animal model for human development, physiology and disease, neurons containing the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT), were mapped in the medulla oblongata of neonatal swine as a first step in identifying their roles in central autonomic control. Neurons were labeled immunocytochemically by using an antiserum to PNMT raised in rabbits against trypsin-treated enzyme purified from the bovine adrenal gland. The general regional organization of neurons expressing PNMT (-like) immunoreactivity (ir) in the neonatal swine was similar to data obtained in other species and, in some aspects, more closely resembled the pattern observed in the primate brain. Immunolabeled cells appeared to be more abundant and caudally more extensive than observed in other adult animals. PNMT-immunoreactive (ir) neuronal somata, however, were largely confined to the reticular formation in the ventrolateral quadrant and the nucleus tractus solitarii (NTS) and more restricted in distribution than those expressing tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (D beta H)-ir on serial transverse sections. A close correspondence was observed between the distributions of TH- and PNMT-ir neurons and processes throughout the C1 and C2 areas. However, in the C1 and C3 regions TH-ir neurons outnumbered those containing D beta H and PNMT-ir. In contrast, cell groups enriched in PNMT-ir neurons and processes were characterized by relatively weak D beta H-ir. In the ventrolateral medulla (VLM), PNMT-ir cell bodies were concentrated rostrally and extended from the caudal pole of the facial nucleus to a level posterior to the calamus scriptorius. The rostral VLM was characterized by an admixture of bipolar and multipolar primarily medium-diameter immunostained neurons. A prominent cell column (condensation) organized ventromedially to the nucleus ambiguus pars compactus (NAc). A loosely organized cluster bordered the lateral aspect of the special visceral efferent column; another smaller aggregate was located in the ventromedial reticular formation adjacent to the inferior olive. At middle medullary levels, PNMT-ir neurons formed two distinct subgroups (dorsal and ventral) interrupted by a band of precerebellar relay neurons that extended between the medial and lateral limbs of the lateral reticular nucleus of Walberg. At obex, the dorsal cell group formed a diagonal array and assumed a position dorsal and dorsolateral to the medial limb of LRN. This group was distinguished by bipolar neurons with axes of orientation directed perpendicularly to the majority of neurons in the rostal VLM or those lying near the caudal ventromedullary surface.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Presumptive adrenergic neurons containing phenylethanolamine N-methyltransferase immunoreactivity in the medulla oblongata of neonatal swine. 135 61

In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no study on the effects of proteases on purified tyrosinase has been reported. We have purified the melanosomal and cytosolic tyrosinases from B16 mouse melanoma and analyzed their susceptibility to trypsin digestion. Both isoforms are sensitive to trypsin, and display similar peptide maps and kinetics of proteolysis, suggesting that they are products of the same gene. The peptide maps and the kinetics of appearance of the fragments were consistent with the sequential removal of N-terminal peptides, leading to a core of 55.3 kDa for the melanosomal form and 48.6 kDa for the cytosolic enzyme. This core was apparently resistant to further proteolysis and catalytically inactive. The difference in molecular weight for the core of the cytosolic and melanosomal forms is the same as that calculated for the native isoforms. The kinetics of enzyme inactivation indicate that the tyrosine hydroxylase and Dopa oxidase activities of tyrosinase are lost at the same rate, and should therefore display similar if not identical structural requirements. The results are discussed in terms of the relationship of both isoforms and of the putative protein sequences deduced from the cDNA clones proposed for tyrosinase.
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PMID:Proteolysis with trypsin of mammalian tyrosinase isoforms from B16 mouse melanoma. 149 41

Expression of tyrosine hydroxylase (TH) in cultured cells of the ventral hypothalamus-midbrain of fetal rats has been investigated. TH mRNA and TH were quantified by an S1 nuclease protection assay and an immunoblot assay, respectively. Dihydroxyphenylalanine (DOPA) and dopamine secretion were evaluated using their rates of accumulation in the culture medium. The rate of accumulation of DOPA was 2-3 times that of dopamine. Inhibitors of TH activity caused a dose-dependent reduction in DOPA secretion. During an 11-week culture of dissociated cells, TH mRNA increased from 1.6 to 2.8 attomole/well between the first and fourth week of culture, remained steady to the ninth week, and then declined. TH increased from 12 to 105 fmol/well between the first and seventh week and then declined. DOPA secretion increased until the sixth week and then remained steady to the tenth week. An extract of rat pituitaries stimulated DOPA secretion by the cultures in a dose-dependent manner. This activity, attributed to a cytotropic factor (CTF), was inactivated by heating for 10 min in a boiling water bath, but was unaffected by trypsin digestion. Incubation with CTF for 24, 48, 72, and 96 h resulted in a day by day increase in the secretion of DOPA. After 96 h of culture with CTF, the amount per well of TH mRNA, but not TH, was significantly (P less than 0.01) greater than the control value. Pituitary CTF is probably not PRL, since rat PRL did not appreciably affect DOPA secretion or the amount of TH mRNA or TH in the cells. Withdrawal of CTF from CTF-stimulated cells resulted in a marked reduction in DOPA secretion as well as a decrease in TH mRNA. These results support the hypothesis that the pituitary gland contains a cytotropic factor that stimulates TH expression in fetal brain cells of the hypothalamus-midbrain.
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PMID:Expression of tyrosine hydroxylase in cultured brain cells: stimulation with an extractable pituitary cytotropic factor. 197 Feb 92

The site specificity of tyrosine hydroxylase phosphorylation in intact PC12 cells, labeled with 32Pi, was investigated. Digestion of 32P-tyrosine hydroxylase with trypsin produced five distinct 32P-labeled peptides (termed PC-1 through PC-5). Sequencing of the peptides revealed four acceptor sites: Ser8, Ser19, Ser31, and Ser40. The phosphorylation site in peptides PC-1 (AV-SEQDAK) and PC-2 (RAVSEQDAK) was identified as Ser19. Agents which cause calcium influx increased 32P incorporation into tyrosine hydroxylase at Ser19. PC-3 was identified as QAEAVTSPR, which contains the phosphorylation site Ser31. Nerve growth factor and phorbol dibutyrate increased 32P incorporation into Ser31. PC-4 was identified as the N-terminal amino acid sequence ((M)PTPSAPSPQPK), and the 32P incorporation occurred at Ser8. Of the agents tested, only okadaic acid (a protein phosphatase inhibitor) increased the phosphorylation of Ser8. PC-5 was shown to contain Ser40. Treatment of the PC12 cells with cAMP-acting agents increased 32P incorporation into Ser40. The present results demonstrate that some, but not all, of the phosphorylation sites demonstrated previously in vitro exist in situ. Conversely, the identification of Ser31 establishes a physiological phosphorylation site not previously reported in vitro. These four sites account for most, if not all, of the diversity in tryptic phosphopeptides reported previously for rat tyrosine hydroxylase.
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PMID:Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40. 197 63

Dopaminergic neurons of the A 10 cell group in the rat ventral tegmental area (VTA) exhibit electrical and dye coupling. Also, the activity of these neurons at least partially reflects their content of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. We examined the ultrastructural localization of TH to determine the morphological features of dopaminergic neurons in the VTA and the relationships between their TH immunoreactivity content and afferent input. Antiserum against the trypsin-treated form of TH was localized using peroxidase-antiperoxidase (PAP) and immunoautoradiographic methods. Immunoreactivity was detected in perikarya, dendrites, and terminals. The perikarya contained the usual organelles, as well as cilia, lamellar bodies, and subsurface cisterns. Qualitative evaluation of peroxidase reaction product and quantitative analysis of the number of silver grains/unit area revealed varying amounts of TH immunoreactivity in nuclei and cytoplasm. Lightly or intensely labeled nuclei were not necessarily associated with corresponding cytoplasmic labeling density. However, cytoplasmic labeling directly corresponded to the relative frequencies of neuronal appositions and synaptic input. Those neurons with less dense cytoplasmic PAP product received fewer synaptic contacts and were less frequently in apposition to other TH-labeled soma and dendrites than neurons displaying relatively more dense cytoplasmic PAP product. Analysis of single sections revealed that 67% (n = 71) of all TH-labeled somata and 15% (n = 2431) of all TH-labeled dendrites were in apposition to other TH-labeled soma or dendrites. TH-labeled terminals were rarely detected and contained relatively low levels of immunoreactivity. The majority of labeled terminals (n = 29/46) formed synapses with labeled soma and dendrites. Unlabeled terminals (n = 2424) in contact with TH-labeled dendrites appeared to form predominantly symmetric synapses. Ten percent (n = 248) of the unlabeled terminals dually synapsed onto adjacent immunoreactive dendrites, perikarya, or dendrite and perikaryon. We conclude that in the rat VTA, (1) detected TH immunoreactivity in cytoplasm, but not nucleus, corresponds to the level of feedback principally from nondopaminergic afferents; (2) dendrodendritic as well as axodendritic synapses between TH-immunoreactive neurons may mediate dopaminergic autoinhibition; and (3) gap junction-like appositions between neurons and convergent inputs from unlabeled terminals onto TH-immunoreactive profiles provide an anatomical substrate whereby cellular activities might be coordinated under certain conditions.
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PMID:Ultrastructural localization of tyrosine hydroxylase in the rat ventral tegmental area: relationship between immunolabeling density and neuronal associations. 197 39

In order to maintain a chronic supply of growth factor for medulla cells in vitro, chromaffin cells from rat, African green monkeys and man were co-cultured with C6 glioma cells, which secrete growth factors that sustain sympathetic neurons in vitro. The response of chromaffin cells to coculture was compared to treatment of medullary cells with nerve growth factor (NGF) alone. Dispersed chromaffin cell preparations were obtained by a trypsin-collagenase procedure, and subjected to differential plating on collagen-coated surfaces. With both human and monkey tissue, non-chromaffin cells did attach to the culture plates and an enriched chromaffin cell population could be replated. Rat adrenal medulla cells survived very poorly in vitro and were not enriched in this procedure. Cultured human and monkey chromaffin cells survived as epithelial cells (50%) and showed neuritic outgrowth on 55 to 66% of the cells after eight days when treated with nerve growth factor (NGF). These cells showed strong catecholamine histofluorescence, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) immunoreactivity. In contrast, only ten percent of adult rat chromaffin cells survived in culture, although NGF treatment rescued an additional 20% of the cells and induced neuritic outgrowth after one week in vitro. C6 glioma cells were treated with mitomycin C bromodeoxyuridine to inhibit mitosis and were plated with the various medulla cells in a one to one ratio. Both human and monkey chromaffin cells expressed extensive and enhanced neuritic arborization within eight days of co-culture, (64-82% respectively) and exhibited intimate contact with the glioma cells as seen at the ultrastructural level. Importantly, survival of adult rat adrenal medulla cells was enhanced to 50% or more with 40% of the cells extending neurites when co-cultured with glioma cells for seven days. Chromaffin cells from all three species reacted for TH, DBH and PNMT in co-culture and were histo-fluorescent. The majority of these cells were also immunoreactive for serotonin and enkephalin, while only 37% of chromaffin cells indicated the presence of NPY. These data indicate that adrenal medulla can be maintained in vitro as the neuronal phenotype when co-cultured with growth factor producing cells and that this strategy may be useful for in vivo transplantation studies.
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PMID:Rodent and primate adrenal medullary cells in vitro: phenotypic plasticity in response to coculture with C6 glioma cells or NGF. 256 44

Chromaffin cells from the monkey adrenal medulla were maintained in vitro in the presence of nerve growth factor (NGF) and the neuronal properties of these cells were assessed. Single-cell preparations were obtained by collagenase-trypsin treatment of the minced adrenal medulla tissue. Cells assumed a glandular to epithelioid morphology after twenty-four hours of culture. Twelve percent of these cells were shown to extend neurites spontaneously after five days. NGF-stimulated neuritic outgrowth from most cells after five days of culture and these neurites remained for at least three weeks. Cells exhibited intense histofluorescence for catecholamines even after three weeks in vitro in the presence of NGF and positive staining for tyrosine hydroxylase and dopamine beta hydroxylase could be detected by immunocytochemistry. Moreover, the chromaffin cells were shown to bind tetanus toxin, which is a specific marker for neurons. Tetanus toxin labelling was not dependent upon the presence of neurites on these cells. Transmission electron microscopy indicated that cultured cells contained numerous dense-core vesicles similar to non-cultured medulla cells. Many of the neurites possessed the morphological features of axons; long varicose processes resembling noradrenergic fibers were identified by catecholamine histofluorescence and tyrosine hydroxylase immunocytochemistry. Microtubular arrays, in an axonal-like organization pattern, were seen ultrastructurally along with the presence of many dense-core vesicles. These data support the potential of adult primate chromaffin cells as a source of sympathetic neuronal tissue for neural transplantation.
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PMID:Neuronal properties of monkey adrenal medulla in vitro. 287 Aug 11

As a first step towards the identification and purification of the molecule(s) that are involved in cell contact-mediated tyrosine hydroxylase (TH) induction in cultures of bovine adrenal chromaffin cells, we have prepared plasma membranes (PM) from bovine adrenal medulla and tested their ability to mimick cell contact-mediated TH induction in low density chromaffin cultures. PM indeed induced TH in a manner similar to that observed in high density cultures. The maximal TH induction reached by PM corresponded to 69% of that of high density cultures, and half-maximal TH induction was obtained with 12 micrograms of PM per ml of medium. The induction of TH by PM was blocked by alpha-amanitin as observed in high density cultures. Since acetylcholinesterase was neither induced in high density nor in PM-treated low density cultures, an induction of TH as a result of a general increase in protein synthesis was excluded. The cell contact molecule(s) appear to be intrinsic membrane proteins. They were not removed by high or low salt extraction, but solubilized by 50 mM octylglucoside. They were resistant to 0.1% trypsin and heat denaturation but inactivated by 0.01% chymotrypsin. PM isolated from the adrenal cortex, kidney, and liver also induced TH in low density chromaffin cell cultures, although to a smaller extent than PM of the adrenal medulla. In contrast, muscle and erythrocyte PM were inactive. This shows that the cell contact molecule(s) are not restricted to the adrenal medulla, but are also present in some other but not all tissues.
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PMID:Selective induction of tyrosine hydroxylase by cell-cell contact in bovine adrenal chromaffin cells is mimicked by plasma membranes. 287 96

Tyrosine hydroxylase, a key enzyme in the biosynthesis of catecholamines, was previously shown to be phosphorylated on four distinct serine residues in PC12 cell cultures, each one being specific for the kinase system involved (McTigue, M., Cremins, J., and Halegoua, S. (1985) J. Biol. Chem. 260, 9047-9056). A cAMP- and Ca2+-independent protein kinase was found to be associated with tyrosine hydroxylase purified from rat pheochromocytoma tumor. The use of this activity and the availability of a large amount of purified tyrosine hydroxylase allowed identification of the site phosphorylated by this kinase activity. A peptide of 1.5 kDa (about 12 residues long), carrying the phosphorylation site, was released from 32P-labeled tyrosine hydroxylase by limited proteolysis with trypsin. This peptide was isolated from trypsinized tyrosine hydroxylase by sequential gel filtration and ion exchange chromatographies. Analysis by thin layer chromatography of an acid hydrolysate of the peptide revealed that it contained phosphoserine. The sequence determination of the peptide showed that it corresponded to the residues 38-45 in the tyrosine hydroxylase primary structure (Arg-Gln-Ser(P)-Leu-Ile-Glu-Asp-Ala). Thus, the associated kinase phosphorylated Ser-40, one of the phosphorylation sites for the cAMP-dependent protein kinase also found in rat pheochromocytoma tumors. These results are compared to those recently appearing in a report by Campbell et al. (Campbell, D. G., Hardie, D. G., and Vulliet, P. R. (1986) J. Biol. Chem. 261, 10489-10492).
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PMID:Rat pheochromocytoma tyrosine hydroxylase is phosphorylated on serine 40 by an associated protein kinase. 288 82

Mild trypsin proteolysis of tyrosine hydroxylase (TH) produces a 34 kDa fragment which is catalytically active. To determine the structure of the trypsin-digested tyrosine hydroxylase (tTH) relative to the native enzyme and to regulatory phosphorylation sites, bovine adrenal tTH was purified to homogeneity and the sequence of 17 amino acids from the N-terminus was determined. These data indicate that the N-terminus of tTH corresponds to amino acid 158. Thus the catalytic region is contained within the central region of enzyme approximately 17 kDa from the N-terminal and 5 kDa from the C-terminal and does not include phosphorylation sites located in the N-terminus. This region of TH shares a high degree of homology with phenylalanine hydroxylase and tryptophan hydroxylase and thus reflects a selective conservation of regions required for catalysis in contrast to the non-homologous regulatory sites. Activation by proteolysis corresponds to an increase in affinity for both substrate and cofactor indicating that the region removed by proteolysis imposes additional constraints on substrate and cofactor binding. These data are consistent with the model that the catalytic core of TH is contained within a 34 kDa region in the highly conserved central portion of the molecule whereas the non-homologous N-terminus regulates cofactor binding and directs substrate specificity.
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PMID:Characterization of the catalytic domain of bovine adrenal tyrosine hydroxylase. 289 48


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