IUB BIOL-L 211 - Signal Transduction (4 pages)

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Signal Transduction

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Signal Transduction


Signal Transduction and Intro- protein detection

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Indiana University, Bloomington
Biol-L 211 - Molecular Biology
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BIOL L211 Lecture 34 Outline of Last Lecture I Antibiotics II Article III Protein Folding IV Protein Targeting Eukaryotes Outline of Current Lecture I Article II Signal Transduction III Protein detection Current Lecture Signal Transduction I Article A Does Turkey Make You Sleepy Scientific American B Tryptophan stands accused of causing post turkey sleepiness 1 An amino acid found in turkey 2 Used to make serotonin a Serotonin sleep regulating neurotransmitter 3 But competitive inhibition negates the effect of added tryptophan a Turkey has other amino acids in greater quantities than tryptophan C Carbohydrates may actually be the guilty party 1 Stimulate the release of insulin These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute 2 Insulin does not directly help tryptophan access the brain but it reduces competition by increasing the uptake of other amino acids into the muscles out of the blood II Signal Transduction A Method of intercellular communication 1 Transduction Cascade series of biochemical reactions that ultimately reaches a target and causes some type of cellular response series of 2 Signal Transduction Cascade some signal interacts with a cell surface receptor which activates intermediate molecules most commonly a phosphorylations 3 Typical cellular response triggered by the end of the signal transduction cascade is the activation or repression of a particular gene B Ligand 1 Ligand the signal that binds to the cell surface receptor 2 Can be hormone sugar protein etc 3 Source and target of the ligand changes signaling classification a Paracrine cells release signals into the extracellular medium and the signals act on local targets only signaling b Juxtacrine signals are tethered to the cell membrane rather than secreted these can only signal to cells in direct contact with the cell juxta next to can only signal to cells next to it c Endocrine cells release signals into the bloodstream that act on distant target cells ex hormones d Autocrine a cell secretes a signal that stimulates itself by binding to that cell s surface receptors C Cell Surface Receptor 1 Has extracellular domain that is bound by the ligand 2 Has intracellular domain that relays the signal by beginning a signal transduction cascade 3 The receptor responds to a ligand by either changing shape or coming in contact with one or more receptors ex dimerizing 4 Receptor Tyrosine Kinases RTK enzyme that phosphorylates a protein a Common cell surface receptor b Dimerizes in response to a ligand which activates the kinase domains c Activated RTKs phosphorylate each other on the tyrosine amino acid hence Receptor Tyrosine Kinase d The signal pathway is then active D MAPK Pathway 1 Mitogen activated protein kinase MAPK pathway a Mitogen binds RTK which dimerizes and trans phosphorylates b Grb2 binds the phosphorylated RTK and SOS which then binds Ras which activates Ras by exchanging GDP for GTP and changing its conformation c Ras activates kinase MAPKKK activation occurs by phosphorylation d MAPKKK activates MAPKK e MAPKK activates MAPK f MAPK activates transcriptional activators which enter the nucleus to activate transcription eukaryotes III Protein Detection A Proteins have a greater variety than DNA meaning a wider variety of procedures is necessary to study them B Separation of proteins on polyacrylamide gels 1 Proteins do not have uniform charge or structure and the structures can be simple or complex 2 Sodium Dodecyl Sulfate SDS serves to give proteins a uniform negative charge which denatures them a SDS is a strong ionic detergent 3 Beta mercaptoethanol BME reduces disulfide bonds a Disulfide bonds are found between cysteine residues C SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis 1 SDS and BME denature proteins and give them a uniform negative charge 2 A stain non specifically binds proteins and permits visualization 3 An electrical current separates proteins by size as they all have uniform charge on a polyacrylamide gel D Western blot visualization technique performed after SDS PAGE 1 Allows visualization of a specific protein 2 After SDS PAGE proteins are transferred to a membrane that nonspecifically binds proteins 3 The proteins maintain their separated positions on the membrane 4 The remaining nonspecific binding sites are blocked a Powdered milk solution is often used 5 The membrane is incubated with a solution of an antibody specific to the desired protein 6 The antibody protein complex is visualized with an enzyme that binds the antibody and releases light

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