Lecture 4 and 5 Cell and molecular endo w10

Lecture 4 and 5 Cell and molecular endo w10 - Lectures 4...

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Lectures 4 and 5 Cellular and Molecular Endocrinology Part I I. General properties of hormone receptors -Methods to study hormone receptor properties adioligand binding assay Radioligand binding assay Receptor expression: Western/Northern blot Immunohistochemistry/In situ hybridization II. How lipophilic hormones work -Puffing in Drosophila polytene chromosomes -Estrogen control of vitellogenesis -Transient transfection assay to identify hormone yy response elements III. Anatomy of a nuclear hormone receptor i t tf t i t d t t t -Transient transfection assay to demonstrate receptor activity and map receptor functional domains IV. Current models of nuclear receptor action -Steroid hormone receptors -RXR heterodimers
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Hormones act through receptors: I. General properties of hormone receptors Bind hormone tightly and specifically (high affinity, low capacity) Induce biochemical chain of events in response to hormone Expressed in target cells Lipophilic hormones Cholesterol derivatives, Fat soluble vitamins, hyroid hormone Hydrophilic hormones Peptides, amino acid derivatives Thyroid hormone R [Second messengers] Protein phosphorylation R ranscription Transcription Transcription
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Determining the affinity of hormone for receptor and how many binding sites (i.e. receptors) are present in the sample The radioligand binding assay gg y Set 1: Constant receptor, add increasing radiolabeled hormone otal) (Total) Set 2: Constant receptor, add increasing radiolabled hormone along with constant saturating nonradiolabeled hormone (Nonspecific)
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Radioactive (hot) hormone onradioactive (cold) hormone Nonradioactive (cold) hormone Receptors bound to support o determine Total binding: To determine Total binding: Detect =1 Detect =4 Detect =8 Low “hot” hormone dded Medium “hot” hormone dded High “hot” hormone added Specific=1 Nonspecific = 0 added Specific= 2 Nonspecific= 2 added Specific= 4 Nonspecific =4
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Radioactive (hot) hormone onradioactive (cold) hormone Receptors bound to support Nonradioactive (cold) hormone To determine nonspecific binding: etect=4 Detect=0 Detect=2 Detect=4 Low “hot” hormone added + excess “cold” Medium “hot” hormone added + excess “cold” High “hot” hormone added + excess “cold” Specific=0 Nonspecific = 0 Specific= 0 Nonspecific= 2 Specific= 0 Nonspecific= 4
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Total Bound Specific Total – nonspecific= Specifically bound ormone H Non- ecific hormone Hormone added specific
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Hormone – receptor interactions follow the law of mass action R=receptor =hormone R + H R H H=hormone k 1 = association rate constant ( usually diffusion limited ) k 2 = dissociation rate constant k 1 k 2 If binding reaction is allowed to proceed to equilibrium, the equilibrium dissociation constant (K d ) = = he equilibrium association constant (K is the inverse of this) k 1 k 2 R H [ ] [ R ][ H ] (Eq. 1) (the equilibrium association constant (K a
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Lecture 4 and 5 Cell and molecular endo w10 - Lectures 4...

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