BIOL-L211 Lecture 34 Outline of Last Lecture I. AntibioticsII. ArticleIII. Protein FoldingIV. Protein Targeting (Eukaryotes)Outline of Current LectureI. ArticleII. Signal TransductionIII. Protein detectionCurrent LectureSignal TransductionI. ArticleA. "Does Turkey Make You Sleepy?" –Scientific AmericanB. Tryptophan: stands accused of causing post-turkey sleepiness1. An amino acid found in turkey2. Used to make serotonina. Serotonin: sleep-regulating neurotransmitter3. But competitive inhibition negates the effect of added tryptophana. Turkey has other amino acids in greater quantities than tryptophanC. Carbohydrates: may actually be the guilty party1. Stimulate the release of insulinThese 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 TransductionA. Method of intercellular communication1. "Transduction Cascade": series of biochemical reactions that ultimately reaches a target and causes some type of cellular response2. Signal Transduction Cascade: some signal interacts with a cell-surface receptor, which activates intermediate molecules; most commonly a series of phosphorylations3. Typical cellular response triggered by the end of the signal transduction cascade is the activation or repression of a particular geneB. Ligand1. Ligand: the signal that binds to the cell surface receptor2. Can be hormone, sugar, protein, etc.3. Source and target of the ligand changes signaling classificationa. Paracrine: cells release signals into the extracellular medium and the signals act on local targets onlyb. Juxtacrine: signals are tethered to the cell membrane rather than secreted; these can only signal to cells in direct contact with the signaling cell (juxta = next to; can only signal to cells next to it)c. Endocrine: cells release signals into the bloodstream that act on distanttarget cells (ex: hormones)d. Autocrine: a cell secretes a signal that stimulates itself by binding to that cell's surface receptorsC. Cell Surface Receptor1. Has extracellular domain that is bound by the ligand2. Has intracellular domain that relays the signal by beginning a signal transduction cascade3. 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 proteina. Common cell surface receptorb. Dimerizes in response to a ligand, which activates the kinase domainsc. Activated RTKs phosphorylate each other on the tyrosine amino acid*hence Receptor Tyrosine Kinased. The signal pathway is then activeD. MAPK Pathway1. Mitogen-activated protein kinase (MAPK) pathwaya. Mitogen binds RTK, which dimerizes and trans phosphorylatesb. Grb2 binds the phosphorylated RTK and SOS, which then binds Ras, which activates Ras by exchanging GDP for GTP and changing its conformationc. Ras activates kinase MAPKKK (activation occurs by phosphorylation)d. MAPKKK activates MAPKKe. MAPKK activates MAPKf. MAPK activates transcriptional activators, which enter the nucleus to activate transcription (eukaryotes)III. Protein DetectionA. Proteins have a greater variety than DNA, meaning a wider variety of procedures is necessary to study themB. Separation of proteins on polyacrylamide gels1. Proteins do not have uniform charge or structure, and the structures can be simple or complex2. Sodium Dodecyl Sulfate (SDS) serves to give proteins a uniform, negative charge, which denatures thema. SDS is a strong ionic detergent3. Beta-mercaptoethanol (BME): reduces disulfide bondsa. Disulfide bonds are found between cysteine residues)C. SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis1. SDS and BME denature proteins and give them a uniform negative charge2. A stain non-specifically binds proteins and permits visualization3. An electrical current separates proteins by size (as they all have uniform charge) on a polyacrylamide gelD. Western blot: visualization technique performed after SDS-PAGE1. Allows visualization of a specific protein2. After SDS-PAGE, proteins are transferred to a membrane that nonspecifically binds proteins3. The proteins maintain their separated positions on the membrane4. The remaining nonspecific binding sites are blockeda. Powdered milk solution is often used5. The membrane is incubated with a solution of an antibody specific to the desired protein6. The antibody-protein complex is visualized with an enzyme that binds the antibody and releases
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