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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ovarian and Leydig cell LH/hCG receptors purified to homogeneity were identified as a single protein of Mr 80,000 and 90,000 respectively. The homogeneity of this protein was confirmed by microsequencing of the first 18 amino acids of the ovarian receptor. The unblocked N-terminal peptide consisted of NH2-R-E-L-S-G-S-R-X-P-E-P-X-D-X-A-P-D-G. These receptors are N-linked sialoglycoproteins which accounts for the size difference between testicular and ovarian receptors and may participate in the interaction with gonadotropin. Crosslinking of pure receptor with hCG with 125I label in either subunit indicated significant interaction of alpha-hCG with the receptor, while beta-hCG seems involved mostly through association and conformational influence on the alpha-subunit. Comparison of Mr derived from SDS with those from FPLC suggested that the native LH receptor are dimers of identical subunits. Autoradiographs of blotted receptors demonstrated that both monomeric and dimeric forms can bind hCG. Receptors from both tissues can be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase and phosphopeptide maps were identical. Receptor occupancy by agonist leads to a conformational change which facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to gonadotropin. Aggregation or dimerization of the hCG/LH receptors could promote clustering and or crosslinking of receptors in the membrane favouring the initial transduction steps in the action of these hormones.
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PMID:Characterization and structure of ovarian and testicular LH/hCG receptors. 260 25

Purified rat Leydig cell cytosol was found to contain a protein kinase which was dependent on the presence of both calcium and phospholipids (phosphatidylserine and diolein), i.e. calcium/phospholipid-dependent protein kinase. The peak of Ca/phospholipid-dependent protein kinase was separated from type I and type II cAMP-dependent protein kinase by DE-52 chromatography. 4 beta-Phorbol-12-myristate-13-acetate (PMA), a tumor-promoting agent, could substitute for diolein in activation of Ca/phospholipid-dependent protein kinase. PMA caused dose-dependent increments of testosterone formation by Leydig cells, whereas inactive phorbol esters had no significant effects. PMA-induced testosterone formation was dependent on extracellular calcium and could be blocked by the addition of the calcium channel-blocking agent nifedipine. Since PMA can directly activate Ca/phospholipid-dependent protein kinase and increase testosterone formation, these results suggest that Ca/phospholipid-dependent protein kinase may be involved in modulating Leydig cell steroidogenesis in addition to the classical cAMP-dependent protein kinase pathway.
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PMID:The role of calcium/phospholipid-dependent protein kinase in Leydig cell steroidogenesis. 298 10

When the phorbol ester, 4 beta-phorbol-12-myristate-13-acetate (PMA) or bacterial phospholipase C (PL-C) is added to a preparation of purified adult rat Leydig cells, containing 2 mM CaCl2, a time- and dose-dependent decreases of LH-stimulated testosterone production is observed. After a 3 h stimulation with oLH (100 ng/ml), PMA (100 ng/ml) and PL-C (1.6 U/ml) do not affect the cell viability or the hCG specific binding, while cAMP accumulation is significantly reduced; cAMP-stimulated steroidogenesis is diminished only in the presence of PL-C. These observations suggest that in vitro: (i) activated Ca2+- and phospholipid-dependent protein kinase is implicated in the regulation of rat Leydig cell steroidogenesis by LH at a step before the adenylate cyclase; (ii) phospholipids play an important role in cAMP-stimulated testosterone synthesis.
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PMID:Effect of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells. 299 25

Gossypol, a phenolic compound that has been studied as a potential male contraceptive, inhibits basal and LH-stimulated testosterone release from Leydig cells in vitro. The present study investigates the mechanism of this inhibition using preparations of purified mouse Leydig cells. Gossypol inhibited LH-stimulated progesterone, 17-hydroxyprogesterone and androstenedione production by mouse Leydig cells incubated in vitro. It also inhibited dibutyryl cyclic AMP-stimulated production of testosterone but was without effect in the presence of added pregnenolone or 25-hydroxycholesterol. These results suggest gossypol exerts its major inhibitory effect on Leydig cell function at a point between LH-dependent stimulation of cyclic AMP dependent protein kinase activity and increased availability of cholesterol for side chain cleavage.
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PMID:Site of gossypol inhibition of steroidogenesis in purified mouse Leydig cells. 302 16

Activation and regulation of Leydig cell function is exerted primarily by LH, which is secreted in pulses of high biological activity and interacts with membrane receptors. Other hormones and factors secreted by the Leydig cell or from the tubular compartment can influence Leydig cell differentiation and acute or chronic actions of LH on steroidogenesis. Conversely, hormones produced in the Leydig cell could modulate tubular function (e.g. beta-endorphin, oxcytocin). The LH receptor has been purified to homogeneity in sufficient quantities to allow its peptide sequence to be determined and its gene structure to be elucidated as well as functional reconstitution studies to be performed. The LH receptor subunit of Mr 90,000 can be phosphorylated by cAMP-dependent protein kinase. The native receptor appears to exist in the membrane as a dimer of identical subunits associated by noncovalent interactions. It is likely that receptor dimerization and further aggregation are necessary for signal transduction to occur, and receptor phosphorylation by one or more kinases may be involved in regulating gonadotropin action. Stimulation of the androgen pathway occurs mainly through a cAMP-mediated mechanism. The stimulatory event can be negatively influenced by the action of certain peptide hormones through the guanyl nucleotide inhibitory subunit of adenylate cyclase. Such an inhibitory action of angiotensin has further emphasized the importance of the cAMP pathway in the Leydig cell. The hormone also appears to facilitate androgen production by a cAMP-independent mechanism located at the plasma membrane or intracellular sites. A Ca2+ sensitive kinase system is present in the Leydig cell membranes. The presence of nM amounts of Ca2+ induces membrane phosphorylation of a protein Mr 45,000. Adenylate cyclase activation also is affected by Ca2+. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH-induced actions in the activated Leydig cell membrane. In the adult rat testis, the ability of Leydig cells to respond to sustained gonadotropic stimulation with increased androgen production is limited by the development of a refractory state associated with loss of LH receptors and steroidogenic enzymes. Gonadotropin-induced steroidogenic lesions in adult rat testes include a late steroidogenic lesion at the site of conversion of progesterone to androgen and an early lesion before pregnenolone formation that leads to a decreased in vitro pregnenolone and testosterone response to hCG.
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PMID:Endocrine regulation and communicating functions of the Leydig cell. 328 2

We have purified the testicular luteinizing hormone (LH/human choriogonadotropin (hCG)) receptor by sequential affinity chromatography on hCG-Sepharose. The purified LH/hCG receptor was identified as a single protein of Mr = 90,000 +/- 2,000 on sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), showed high affinity binding for hCG, and a binding capacity of 3.8 nmol/mg of protein. Electrophoretically blotted receptor retained the ability to bind 125I-hCG on nitrocellulose membrane, and the Mr of radioactive band was consistent with that revealed by silver staining. Autoradiography after SDS-PAGE analysis of cross-linked purified receptor-hCG complex showed Mr = 145,000 and Mr = 105,000 bands. These results are consistent with a Mr value for the receptor of 90,000 after accounting for contribution by the intact hormone or its alpha-subunit. Analysis of the free receptor by fast protein liquid chromatography on Superose 12 revealed a single peak of binding activity for 125I-hCG which eluted in the position of Mr = 200,000-240,000 in the presence of Triton X-100. Since a single protein species is observed under reducing or nonreducing conditions in SDS-PAGE, the receptor could exist in the membrane as a dimeric form composed of subunits Mr = 90,000 associated through noncovalent interactions. The pure receptor can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase (approximately 0.3 mol of phosphate/mol of receptor). This phosphorylation does not affect the binding characteristics of the receptor. The method described is simple and allows rapid purification of microgram amounts of biological active Leydig cell LH/hCG receptor for structural, functional, and immunological studies.
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PMID:Purification and characterization of Leydig cell luteinizing hormone receptor. 368 Feb 91

Gonadotropin-releasing hormone agonist (GnRHa) markedly increased testosterone formation from 2.35 +/- 0.13 ng/ml of the controls to 14.92 +/- 0.33 ng/ml (mean +/- SE) in isolated and purified rat Leydig cells. GnRHa-induced testosterone formation was completely blocked by phospholipase A2 inhibitor (chloroquin, 10(-4) M), but was potentiated by the addition of either cyclo-oxygenase inhibitor (indomethacin) or lipoxygenase inhibitor (nordihydroguaiaretic acid, NDGA). Arachidonic acid also directly stimulated Leydig cell steroidogenesis and activated Ca/phospholipid dependent protein kinase. Steroidogenic effects of arachidonic acid were also potentiated by the addition of either indomethacin or NDGA. These results suggest that arachidonic acid may be important in mediating direct stimulatory effects of GnRH on Leydig cell steroidogenesis, and the conversion of arachidonic acid to either prostaglandins or leukotrienes is not required for its steroidogenic effect.
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PMID:Mechanism of action of gonadotropin-releasing hormone stimulated Leydig cell steroidogenesis. III. The role of arachidonic acid and calcium/phospholipid dependent protein kinase. 392 Apr 63

Leydig cell cAMP-dependent protein kinase has been characterized using rapid fractionation and optimal conditions to minimize proteolysis. DEAE-cellulose analysis showed a single Type I peak of cAMP binding and enzyme activity that eluted at 0.1 M KCl. Photoaffinity labelling with 8-azido[32P]cAMP followed by SDS-PAGE showed a doublet with Mr 54000 and 51000 for the peak fraction, while the original extract exhibited only the smaller form. Autophosphorylation revealed a doublet of Mr 54000 +/- 573 and Mr 51000 +/- 710. To titrate the occupancy of regulatory subunits during hCG action, free cAMP receptors were measured by 8-azido[3H]cAMP binding under non-exchange conditions followed by photolysis. hCG treatment caused a dose-related decrease of free receptors and SDS-PAGE analysis of the 8-azido[32P]cAMP regulatory subunit from control and hCG treated cells also showed a hormone dependent decrease in a single band of Mr 50000. These results have shown that the Leydig cell protein kinase behaves as a Type I enzyme on DEAE analysis, but has the physical characteristics of the Type II enzyme. The dose-dependent fall in available receptor sites during hCG stimulation further indicates the central role of cAMP in hormone action in the Leydig cell.
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PMID:Characterization of Leydig cell protein kinase. Further studies in hormone action. 609 57

Epidermal growth factor (EGF) and cyclic AMP were found to stimulate distinct protein kinase activities in plasma membranes prepared from the M5480P murine Leydig cell tumor. EGF stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 180,000, while cyclic AMP stimulated the phosphorylation of a minor component of molecular weight 220,000. The two types of kinases could also be distinguished on the basis of differential susceptibility to conditions of membrane preparation. These results suggest that EGF stimulates a cyclic AMP-independent protein kinase in murine Leydig cell tumors at the level of the plasma membrane.
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PMID:Epidermal growth factor and cyclic AMP stimulation of distinct protein kinase activities in Leydig cell tumor membranes. 628 36

The control of androgen production by the Leydig cell is dependent upon the episodic secretion of hormone (LH), which is released from the anterior pituitary gland in pulses of high biological activity. This mode of episodic LH secretion supports steroidogenic enzyme activity in the testis through interaction with LH receptors and stimulation of the adenylate cyclase/protein kinase sequence, leading to phosphorylation of key intermediates in the steroid biosynthetic pathway. The plasma membrane events that are rapidly activated by the specific interaction of LH or hCG with Leydig cell receptors include increased binding of guanyl nucleotide, and stimulation of cAMP-independent, Ca2+-dependent phosphorylation of a 44,500 Mr protein, with the characteristics of the adenylate cyclase nucleotide regulatory unit. Hormonal activation of adenylate cyclase is affected by Ca2+ with the same concentration-dependence, suggesting that nucleotide-induced phosphorylation is related to activation of the catalytic cyclase unit. In addition to the characteristic increases in pregnenolone synthesis and androgen production, gonadotropin-stimulated Leydig cells show prominent changes in LH receptor content and steroidogenic activity that modify their subsequent responses to hormonal signals. Thus, after exposure to increased LH and hCG levels in vivo and in vitro, LH receptors show an initial transient increase (up-regulation) followed by a marked decrease (down-regulation) and a prolonged depletion of LH receptor sites. Large doses of hCG cause "early" (prior to pregnenolone) and "late" steroidogenic lesions (17 alpha-hydroxylase, 17-20 desmolase) that are independent of receptor loss. The early lesion is partly due to reduced activity of HMG CoA reductase, and is mainly attributable to the increased activity of an inhibitory protein factor that modulates the activity of cholesterol side chain cleavage enzyme in Leydig cell mitochondria. In contrast, the late steroidogenic lesion is related to the nuclear actions of E2 produced during hormonal action. After hCG stimulation, an increase in nuclear E2 binding was accompanied by an early rise of RNA polymerase activities within 45 min coincident with the maximal increases in circulating testosterone and estradiol levels. These events were followed by the emergence of an E2-induced protein of Mr 27,000 at 3-6 h, and by reduction in the activity of 17 alpha-hydroxylase/17-20 desmolase, and a decrease in microsomal cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Hormonal regulation of androgen production by the Leydig cell. 632 62


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