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Tyrosine hydroxylase: purification from PC-12 cells, characterization and production of antibodies.
Tyrosine hydroxylase has been purified to homogeneity from cultured PC-12 cells. The protein migrates as a single band with a molecular weight of 60,000 on sodium dodecyl sulfate polyacrylamide electrophoresis. Two-dimensional electrophoresis of the pure enzyme resolves three spots (every with molecular weights of 60,000) with isoelectric factors of 5.4, 5.Eight and 5.9.
This cost heterogeneity can’t be defined by the presence of sugar or lipid moieties on the enzyme. Amino acid evaluation signifies a comparatively excessive content material of hydrophobic amino acids and a decrease serine content material than different preparations of tyrosine hydroxylase.
The enzyme hydroxylates tryptophan at roughly 1% of its charge of tyrosine hydroxylation however is not going to catalyze the hydroxylation of phenylalanine. Polyclonal antibodies have been produced in rabbits in opposition to pure tyrosine hydroxylase and have been judged to be monospecific by Western blot evaluation.
The IgG fraction was remoted from serum, and when coupled to cyanogen bromide activated Sepharose, may very well be used to purify tyrosine hydroxylase from crude extracts in a single step. The antiserum proved to be very helpful in immunoprecipitation and immunocytochemical experiments with tyrosine hydroxylase.
Function of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis.
The consequences of depolarization by elevated potassium concentrations have been studied in PC12 cells and in stably transfected AtT-20 cells expressing wild-type or [Leu19]-recombinant tyrosine hydroxylase (rTH). Modifications within the phosphorylation states of Ser19 and Ser40 in tyrosine hydroxylase (TH) have been decided immunochemically utilizing antibodies particular for the phosphorylated state of every website and in contrast with adjustments in TH exercise in PC12 cell lysates and with adjustments in L-DOPA biosynthesis charges in intact AtT-20 cells.
Therapy of both PC12 cells or AtT-20 cells expressing wild-type rTH with elevated potassium produced a transient enhance within the phosphorylation state of Ser19 (as much as 0.7 mol of phosphate/mol of subunit) in live performance with a extra gradual and sustained enhance in Ser40 phosphorylation.
Elevated potassium therapy additionally elevated TH exercise in PC12 cell lysates, however these will increase paralleled the temporal course of Ser40, versus Ser19, phosphorylation. Equally, will increase in DOPA accumulation produced by elevated potassium in AtT-20 cells expressing wild-type rTH paralleled the will increase within the phosphorylation state of Ser40 however not Ser19.
Furthermore, elevated potassium produced comparable will increase in DOPA accumulation in AtT-20 cells expressing rTH during which Ser19 phosphorylation had been eradicated (by substitution of Leu for Ser19). Thus, depolarization-induced will increase within the stoichiometry of Ser19 phosphorylation don’t seem to affect immediately the exercise of TH in situ.
Immunohistochemical research on phosphorylation of tyrosine hydroxylase in central catecholamine neurons utilizing site- and phosphorylation state-specific antibodies.
Antibodies raised to phosphorylated types of tyrosine hydroxylase, the primary and rate-limiting enzyme within the catecholamine biosynthesis, have been utilized in immunohistochemical research on rat mind slices incubated in vitro with a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, IBMX) and on forskolin on formalin-perfused rat brains.
4 antisera/antibodies have been used: polyclonal rabbit antisera to (i) tyrosine hydroxylase phosphorylated at serine 40 (THS40p antiserum), (ii) tyrosine hydroxylase phosphorylated at serine 19 (THS19p antiserum), (iii) the native enzyme (pan-tyrosine hydroxylase antiserum), and mouse monoclonal antibody to (iv) native tyrosine hydroxylase.
Within the in vitro research THS40p-like immunoreactivity was not noticed except slices have been handled with IBMX-forskolin after which a dense fibre community was discovered within the striatum, and immunoreactive cell our bodies have been discovered within the ventral mesencephalon, particularly within the ventral tegmental space.
Though these cells have been pan-tyrosine hydroxylase-positive, a number of of them weren’t stained with the tyrosine hydroxylase-monoclonal antibody. Furthermore, there was a marked discount of tyrosine hydroxylase-monoclonal antibody-immunoreactive fibres in drug-treated slices, suggesting that this tyrosine hydroxylase-monoclonal antibody doesn’t acknowledge the serine 40-phosphorylated type of tyrosine hydroxylase.
Handled slices didn’t present any THS40p-immunoreactive cell our bodies within the dopaminergic A11 cell group and only some, weakly fluorescent neurons have been noticed in locus coeruleus. Nevertheless, a sparse fibre plexus was noticed in locus coeruleus, presumably reflecting epinephrine fibres.
Within the perfused brains THS40p-like immunoreactivity may very well be visualized in some dopamine neurons within the ventral mesencephalon, particularly the A10 space, and in noradrenergic locus coeruleus neurons, whereas THS19p-like immunoreactivity was present in all catecholamine teams studied, just like the outcomes obtained with the pan-tyrosine hydroxylase antiserum and the tyrosine hydroxylase-monoclonal antibody.
In forebrain areas recognized to be innervated by mesencephalic dopamine neurons, no THS40p-positive fibres have been noticed, whereas THS19p-immunoreactive fibres have been present in subregions of the striatum, olfactory tubercle and nucleus accumbens, basically overlapping with dopamine fibres beforehand proven to comprise cholecystokinin-like immunoreactivity.
The current outcomes means that antibodies directed in opposition to phosphorylated types of tyrosine hydroxylase can be utilized to guage the state of tyrosine hydroxylase phosphorylation in particular person neuronal cell our bodies and processes each in vitro and in vivo.
Innervation of the rooster parathyroid glands: immunohistochemical examine with the TuJ1, galanin, VIP, substance P, CGRP and tyrosine hydroxylase antibodies.
The innervation of the rooster parathyroid glands was studied by immunohistochemistry utilizing varied antibodies. The parathyroid glands, in addition to the carotid physique and ultimobranchial gland, obtained branches originating from the vagus nerve. Quite a few nerve fibers immunolabeled with the monoclonal antibody (TuJ1) in opposition to neuron-specific class III beta-tubulin isotype have been discovered within the connective tissue capsule and septa penetrating into the parathyroid parenchyma.
They have been additionally outstanding within the wall of blood vessels. Peptidergic nerve fibers immunoreactive for galanin, vasoactive intestinal polypeptide (VIP), substance P and calcitonin gene-related peptide (CGRP) have been densely distributed within the capsule, septa and blood vessel partitions of the parathyroid glands.
As well as, some TuJ1-, substance P- and CGRP-immunoreactive fibers have been detected in shut affiliation with the parenchymal cells of parathyroid glands. Tyrosine hydroxylase-immunoreactive fibers have been concentrated round blood vessels and likewise in connective tissue stroma.
Antibodies to a section of tyrosine hydroxylase phosphorylated at serine 40.
An artificial peptide comparable to residues 32-47 of rat tyrosine hydroxylase (TH) was phosphorylated by protein kinase A at Ser40 and used to generate antibodies in rabbits. Reactivity of the anti-pTH32-47 antibodies with phospho- and dephospho-Ser40 types of TH protein and peptide TH32-47 was in contrast with reactivity of antibodies to nonphosphorylated peptide and to native TH protein. In antibody-capture ELISAs, anti-pTH32-47 was extra reactive with the phospho-TH than with the dephospho-TH varieties.
Conversely, antibodies in opposition to the nonphosphorylated peptide reacted preferentially with the dephospho-TH varieties. In western blots, labeling of the roughly 60-kDa TH band by anti-pTH32-47 was readily detectable in lanes containing protein kinase A-phosphorylated native TH at 10-100 ng/lane. In blots of supernatants ready from striatal synaptosomes, addition of a phosphatase inhibitor was essential to discern labeling of the TH band with anti-pTH32-47.
Equally, anti-pTH32-47 did not immunoprecipitate TH exercise from supernatants ready from untreated tissues, whereas prior therapy with both 8-bromoadenosine 3′,5′-cyclic monophosphate or forskolin enabled removing of TH exercise by anti-pTH32-47. Lastly, in immunohistochemical research, anti-pTH32-47 selectively labeled catecholaminergic cells in tissue sections from perfusion-fixed rat mind.
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