Exam 2 Flashcards
What are the three protein components of the membrane-protein cytoskeleton? What are 2 examples of proteins in each category?
The membrane cytoskeleton is made up of filamentous peripheral membrane proteins linked to transmembrane proteins by proteins that link them together.
Peripheral: Actin and Spectrin
Transmembrane: Band 3 and Glycophorin
Linkers: Ankryin and Band 4.1
What are the three main domains in epithelial cell membranes? How are these domains maintained? What does each domain specialize in?
Apical, lateral, and basal plasma membrane domains. These are separated by tight junctions which keep the specialized proteins in each domain from mixing.
Domain functions:
Apical - nutrient uptake
Lateral - cell-cell communication and adhesion
Basal - generation of ion gradients and transfer nutrients to bloodstream
What is Km and how does it differ in competitive and non-competitive inhibition of enzymes?
Km is “Michaeli’s constant” which represents the substrate concentration at which half of the maximum enzyme rate (Vm) has been reached.
What is the equation for the rate of enzyme function?
Rate = (Vmax * [substrate]) / (Km + [substrate])
What is the procedure for a Wester Blot?
Grind up tissue/cells Solubilize the proteins with detergents Ren un electrophoretic gel Use antibodies raised against protein Label band with these antibodies Look at this "blot" of gel on paper
How does Maldi-Tof Spectroscopy work?
Tissue protein extracts are pro ken into peptides
Mass spec determines they exact molecular weights
Mass spec can figure out which amino acids they are composed of
Mass spec can piece together and identify fragments by matching in database of all know protein sequences
Multiple proteins can be identified
What is the procedure for GFP tagging for protein localization?
Obtain cDNA for the protein by using a reverse transcriptase on the mRNA for the protein of interest to make cDNA. Splice the cDNA for Green Fluorescent Protein (GFP) onto the end of the cDNA for the protein that you make, producing the cDNA for a fluorescent chimeric (multi-gene) protein.Then you can either incorporate the chimeric cDNA into a suitable vector (plasmid or virus) and transfect (transform) a specific cell line with the cDNA for the chimeric protein. The cell line will then express fluorescent X or localize fluorescent X in specific organelle using fluorescence microscopy.
Describe how you would preform in-situ hybridization.
Synthesize a small antisense nucleotide probe that will bind to a short part of the protein’s mRNA sequence. Tag the antisense probe mRNA with an enzyme which creates a colored product – or with a fluorescent dye. Incubate sections with tagged antisense mRNA. Add substrate of tagging enzyme. Look for colored product in the cytoplasm of cells which contain the mRNA for protein X – or use fluorescent microscopy
Describe how you would use a marker enzyme for mRNA discovery.
Sequence genomic DNA for gene and its upstream promoter. Make transgenic animals in which the code for a “marker enzyme” that produces a colored product is inserted next to the gene for the protein of interest, between the protein of interest and its promoter. Add marker enzyme substrate - Look for colored enzyme product in cells expressing the gene.
What are the three types of membrane proteins, and what kinds of bonds attach these proteins to the membrane?
Integral membrane proteins - held in membrane by hydrophobic effect
Lipid-anchored proteins - held my covalent bonds with fatty acid chains in membrane
Peripheral membrane proteins - held by weak bonds to membrane
How would you figure out the rate of protein diffusion in a membrane?
By using FRAP, fluorescence recovery after photobleaching, you can bleach/destroy a section of membrane that has proteins that have fluorescent tags, and with a fluorescent microscope, watch the speed of the recovery of proteins in that area.
Why is channel mediated passive transport faster than transporter mediated?
Because it only takes one conformational change to let many particles through.
What is the channel protein for water? and what is the name for diffusion of water?
Aquaporins are water passive channel proteins, and osmosis refers to the diffusion of water through the membrane
What determines the permeability of a channel?
The probability that the channel is open due to small conformational changes due to “flickering”
What is an example of a passive transporter protein and its function?
GLUT4 is a passive glucose transporter and insulin promotes insertion of GLUT4 into cells to promote glucose uptake.
