Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.1 (chymotrypsin)
 10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine adrenal tyrosine hydroxylase has been obtained in a form that is 85 to 90% pure. Sodium dodecyl sulfate-gel electrophoresis and density gradient centrifugation studies have established that the subunit molecular weight of the chymotrypsin-solubilized enzyme is 34,000. The presence of iron in the purified enzyme (0.50 to 0.75 mol of iron/mol of enzyme) has been established. Crude particulate tyrosine hydroxylase can be activated by the phospholipid, phosphatidyl-L-serine, or by exposure to enzymatic phosphorylating conditions. Both forms of activation lower the Km of the enzyme for its 2-amino-4-hydroxypteridine cofactor. By contrast, tyrosine hydroxylase that has been solubilized by chymotrypsin cannot be activated by either of these methods.
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PMID:Bovine adrenal tyrosine hydroxylase: purification and properties. 1 85

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

Immunocytochemical localization of tyrosine hydroxylase (TH) was performed on paraffin sections pretreated with various proteolytic enzymes. It was found that pretreatment with trypsin (1.2 mg/ml) for 5 min resulted in a dramatic increase in the number of TH-positive terminals throughout the brain, especially in the cerebellum, which contains fine preterminal and terminal axons that are difficult to stain. This pretreatment also led to a significant reduction in background staining and allowed for the use of the TH antiserum at high working dilutions. Several other proteolytic enzymes were tested and only chymotrypsin was nearly as effective as trypsin with respect to TH staining.
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PMID:Optimization of tyrosine hydroxylase immunocytochemistry in paraffin sections using pretreatment with proteolytic enzymes. 614 41

A coordinated series of immunohistochemical and biochemical analyses have been conducted in the hamster to examine the dependence of substance P and tyrosine hydroxylase (TH) expression by second-order olfactory neurons, and the level of dopamine in the main olfactory bulb (MOB), on the integrity of carnosine- and olfactory marker protein (OMP)-containing primary afferent neurons. Substance P-like immunoreactivity (SPLI) is localized in external tufted cells and centrifugal afferents of the MOB; TH immunoreactivity has a wider distribution, in external tufted, middle tufted, periglomerular, and deep short-axon cells as well as in centrifugal afferents. To characterize the SPLI, this material was isolated by guanidine-HCl extraction and passage over a C18 SEP-PAK. The SPLI coelutes on HPLC with authentic substance P and, following oxidation, coelutes with substance P sulfoxide. It is sensitive to alpha-chymotrypsin and is resistant to trypsin. Thus, the SPLI in the MOB behaves as authentic substance P. Intranasal irrigation with 0.17 M ZnSO4 results in peripheral deafferentiation of the MOB for up to 8 months as evidenced by a persistent loss of OMP immunoreactivity and shrinkage of the olfactory nerve layer and glomeruli. By these criteria, the vomeronasal inputs to the accessory olfactory bulb are not destroyed and the spared vomeronasal receptor neurons do not innervate the vacated peripheral projection field in the MOB. The loss of peripheral inputs to the MOB is accompanied by marked and parallel reductions in the incidences of SPLI- and TH-positive second-order neurons despite an increase in the density of neuronal somata in the glomerular layer. Biochemical quantifications following peripheral deafferentation also demonstrate significant decreases of both substance P and dopamine, together with the expected decrease of carnosine. In contrast, the SPLI and the TH and serotoninlike immunoreactivities in centrifugal afferents as well as the TH immunoreactivity in deep interneurons do not appear to be reduced, and the MOB content of norepinephrine in centrifugal afferents is unaffected. These results collectively indicate that the loss of inputs from the primary olfactory receptor neurons can reduce the levels of at least two different, putatively neuroactive compounds (substance P and dopamine) in at least three classes of second-order neurons (external tufted, middle tufted, and periglomerular cells). The control of central neuron phenotype by the peripheral olfactory neurons thus appears to be a phenomenon of broad influence. It may play a role in processing chemosensory information as well as offering a system in which to study neuronal plasticit
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PMID:Substance P and catecholaminergic expression in neurons of the hamster main olfactory bulb. 619 81

In the course of the purification of 14-3-3 protein (14-3-3) we found that 14-3-3 isolated from bovine forebrain activates protein kinase C (PKC), rather than the previously reported protein kinase C inhibitory activity (KCIP). We have characterized the 14-3-3 activation of PKC. The physical properties of purified PKC activator are the same as those previously reported for 14-3-3 and KCIP; i.e., (1) it is composed of subunits of molecular weight 32,000, 30,000, and 29,000; (2) it is homogeneous with respect to molecular weight, as judged by native gradient-gel electrophoresis, with a molecular weight of 53,000; and (3) it is composed of at least six isoforms when analyzed by reverse-phase HPLC. The concentration dependence of PKC activation by 14-3-3 is in the same range as that shown previously for KCIP inhibition of PKC, and as that required for 14-3-3 activation of tyrosine hydroxylase; a maximal stimulation of two- to three-fold occurs at 40-100 micrograms/ml. 14-3-3's activation of PKC is sensitive to alpha-chymotrypsin digestion but is not heat labile. Activation is specific to PKC; at least two other protein kinases, cyclic AMP- and calcium/calmodulin-dependent protein kinases, are not activated. The activation of PKC by 14-3-3 is independent of phosphatidylserine and calcium and, as such, is an alternative mechanism for the activation of PKC that obviates its translocation to membranes.
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PMID:Activation of protein kinase C by purified bovine brain 14-3-3: comparison with tyrosine hydroxylase activation. 793 46

We previously reported that a factor(s) from rat choriocarcinoma (Rcho-1) cells suppresses circulating PRL levels and increases tyrosine hydroxylase activity in tuberoinfundibular dopaminergic neurons in vivo. The purposes of this study were to determine whether this factor(s) increases tyrosine hydroxylase activity in fetal hypothalamic cells in vitro and to evaluate its chemical nature. The Rcho-1 cells are of placental origin and have the capacity to differentiate into giant cells and produce members of the placental PRL family. MMQ cells, a pituitary cell line that secretes PRL, and HRP-1, a placental cell line that does not produce any known members of the PRL family, were used as control cells. Tyrosine hydroxylase activity was assessed by incubation of hypothalamic cells for 1 h with 100 microM brocresine, an inhibitor of aromatic L-amino acid decarboxylase. Tyrosine hydroxylase activity was increased in a density-dependent manner when Rcho-1, but not HRP-1 or MMQ, cells were cocultured with hypothalamic cells for 24 h. Control and Rcho-1-stimulated tyrosine hydroxylase activities were markedly reduced with 1 mM alpha-methyl-p-tyrosine, a specific inhibitor of tyrosine hydroxylase. Tyrosine hydroxylase activity was not altered when hypothalamic cells were incubated for 24 h with rat PRL or recombinant rat placental lactogen-I, whereas a 24-h stimulation with 100,000 Rcho-1 cells and a 1-h stimulation with 5 mM (Bu)2cAMP increased tyrosine hydroxylase activity 3.7- and 3-fold, respectively. The magnitudes of the increase in tyrosine hydroxylase activity were similar when hypothalamic cells were cocultured with Rcho-1 cells for 1 and 24 h. Acetic acid extracts of Rcho-1, but not HRP-1 or MMQ, cells increased tyrosine hydroxylase activity within 1 h in a concentration-dependent manner. The 3-fold increase in tyrosine hydroxylase activity observed with 500,000 Rcho-1 cell equivalents was markedly reduced with 1 mM alpha-methyl-p-tyrosine. The mol wt range of the tyrosine hydroxylase-activating factor(s) (THAF) was estimated using ultrafiltration membranes. The majority of activity was found in the eluate from a 1,000 mol wt cut-off membrane. THAF activity in Rcho-1 cell extracts was decreased by preincubation with pronase, a nonspecific proteolytic enzyme, suggesting that the factor(s) is a peptide. THAF was resistant to inactivation by trypsin or chymotrypsin pretreatment. However, both enzymes destroyed the ability of pituitary adenylate cyclase-activating peptide, either alone or with Rcho-1 cell extracts, to increase tyrosine hydroxylase activity. Oxidation of Rcho-1 cell extracts with performic acid abolished THAF activity.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A factor(s) from a trophoblast cell line increases tyrosine hydroxylase activity in fetal hypothalamic cell cultures. 810 May 18

Tyrosine nitration is a covalent posttranslational protein modification that has been detected under several pathological conditions. This study reports that nitrated proteins are degraded by chymotrypsin and that protein nitration enhances susceptibility to degradation by the proteasome. Chymotrypsin cleaved the peptide bond between nitrated-tyrosine 108 and serine 109 in bovine Cu,Zn superoxide dismutase. However, the rate of chymotryptic cleavage of nitrated peptides was considerably slower than control. In contrast, nitrated bovine Cu,Zn superoxide dismutase was degraded at a rate 1. 8-fold faster than that of control by a gradient-purified 20S/26S proteasome fraction from bovine retina. Exposure of PC12 cells to a nitrating agent resulted in the nitration of tyrosine hydroxylase and a 58 +/- 12.5% decline in the steady-state levels of the protein 4 h after nitration. The steady-state levels of tyrosine hydroxylase were restored by selective inhibition of the proteasome activity with lactacystin. These data indicate that nitration of tyrosine residue(s) in proteins is sufficient to induce an accelerated degradation of the modified proteins by the proteasome and that the proteasome may be critical for the removal of nitrated proteins in vivo.
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PMID:Proteolytic degradation of tyrosine nitrated proteins. 1093 92