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132 Cards in this Set
- Front
- Back
What are the three major isoforms of nitric oxide synthases?
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neuronal NOS (nNOS; NOS I)
inducible NOS (iNOS; NOS II) endothelial NOS (eNOS; NOS III) |
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Which structure does NOT require the substrate to bind to function, P450 or NOS?
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NOS (Arginine does not have to be present for it to synthesis NO)
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What is sGC? Explain the structure. What does it convert?
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Soluble guanylate cyclase.
It is a heterodimer (one alpha, one beta chain), heme-containing protein. Converts GTP to cGMP |
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Where is nNOS found?
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Skeletal muscle and neurons
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What primarily activates NOS I?
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The binding of calcium (via large influx, as in with action potential) to calmodulin, which then binds to nNOS, producing NO
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What activates soluble guanylate cyclase?
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The binding of NO to the heme of sGC, which breaks the His-heme bond.
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Who is responsible for determining the nation's needs and interests?
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Strategists must weigh heavily the nations needs and interests. The process entails determining what interests and what national instruments of power are available and applicable.
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Which NOS isoform produces the most NO?
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iNOS (NOS II)
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Where is P450 found?
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Integral membrane protein
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Where does P450 get its name?
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It is the absorbance that is characteristic when CO is bound
"Pigment 450 nm" |
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Explain the structure of P450
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Very similar to Hb, in that is has the protoporphyrin IX ring, but instead of His being bound, Cys is bound.
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What does NADPH stand for? How many H+ can it transfer at one time?
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Nicotinamide adenine dinucleotide phosphate
TWO |
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What is the general P450 formula?
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NADPH + O2 + H + XH --> NADP+ + H2O + XOH
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Why does the substrate have to be bound in order for the p450 cycle to begin?
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The redox potential of non-bound P450 is -300 mV (low-spin, hexa-coordinated) which is higher than the FMN moiety (-270 mV). Once the substrate is bound, the complex becomes high-spin penta-coordinated, reducing the redox potential to -230, making it capable of accetping electrons.
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What does FAD stand for?
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oxidized flavin adenine dinucleotide.
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What does FADH2 stand for?
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reduced flavin adenine dinucleotide
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What does FMN stand for?
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flavin mononucleotide
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What does FMNH2 stand for?
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reduced flavin mononucleotide
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What structure do FAD and FMN have in common?
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Both contain the isoalloxazine ring from the vitamin B2 (riboflavin)
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What is the active redox group of the flavoprotein?
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Isoalloxazine ring
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Where is the P450 located in the ER? In the mitochondria?
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in the ER: embedded in the cytoplasmic side of the membrane
in the Mitochondria: on the inner membrane |
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NADPH-cytochrome P450 reductase is a_____________ membrane protein which contains which two groups?
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peripheral;
FAD and FMN prosthetic groups |
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Which is in more abundance, NADPH- cytochrome P450 reductase flavoproteins or P450s? what attracts the flavorproteins to the P450?
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NADPH- cytochrome P450 reductase flavoproteins
Electrostatic interactions (once substrate is bound the binding affinity for the flavoproteins is increased) |
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What reactions are mitochondrial P450 involved in ? What is the enzyme that is used?
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steroid hydroxylation reactions
NADPH-adrenodoxin reductase |
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Compare and contrast NADPH- cytochrome P450 reductase flavoproteins and NADPH-adrenodoxin reductase
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NADPH- cytochrome P450 reductase flavoproteins:
Found in ER P450 contains FAD and FMN NADPH-adrenodoxin reductase: found in mitochondria does not contain FMN, but rather 2 adrenodoxin protein to transfer the electrons to P450 |
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Why do we care about P450?
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They affect the amount of time drugs stay in the body.
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How are P450's classified (named)?
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All start with CYP
Family= Number 40% aa sequence identity Subfamily= Letter 50% aa sequence identity Final # given based on the order it was found |
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Why is CYP11A1 important? Where would you find this most often?
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All of the steroids in the body use CYP11A1
Adrenal gland |
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What reaction does CYP11A1 catalyze?
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The committed step in steroid biosynthesis (cleaving the bond between C20 and C22 to form pregnenolone (21 carbon) and isocaproic aldehyde (6 carbon)
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Explain the steps of steroid synthesis
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step 1:
in the adrenal mitochondria, CYP11A1 hydroxylates cholesterol (a 27 carbon) at C22 to produce 22-hydroxycholesterol step 2: CYP11A1 hydroxylate at C20 to produce 20,22-dihydroxycholesterol (a diol) step 3: CYP11A1 cleaves the bond between C20 and C22 to produce pregnenolone (21 C) and isocaproic aldehyde (6 C) |
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Which is the only amino acid that is not chiral?
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Glycine
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All of the biologically active chiral alpha C's have _____________ configuration
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Levorotary
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All the biologically acitve chiral alpha carbons have __________ absolute configuration except __________.
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Sinister;
Cysteine |
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Describe polarity.
Valine |
nonpolar
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Describe polarity.
Leucine |
nonpolar
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Describe polarity.
Isoleucine |
Nonpolar
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Describe polarity.
Phenylalanine |
Nonpolar
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Describe polarity.
Tyrosine |
Nonpolar
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Describe polarity.
Tryptophan |
Nonpolar
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Describe polarity.
Histidine |
Nonpolar
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Describe polarity.
Aspartate |
Polar
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Describe polarity.
Glutamate |
Polar
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Describe polarity.
Lysine |
Polar
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Describe polarity.
Arginine |
Polar
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Describe free energy.
Arginine |
Very large negative free energy
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Describe free energy.
Glutamate |
Very large negative free energy
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Decribe free energy.
Aspartate |
Very large negative free energy
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Describe free energy.
Lysine |
Very large negative free energy
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Describe Free energy.
Tyrosine |
Positive free energy (hydrophobic)
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Describe free energy.
Isoleucine |
Positive free energy (hydrophobic)
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Describe free energy.
Phenylalanine |
Positive free energy
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decribe free energy.
Tryptophan |
Positive free energy
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How would you describe an amino acids with free energy near zero and both hydrophobic and hydrophilic parts?
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Amphoteric
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Two cysteines oxidize to form ___________. What is the name of this bond? Is it covalent or noncovalent?
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Cystine
Disulfide Bond Covalent |
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What is the bond called in involved in polymerization?
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Peptide bond
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What kind of a reaction is polymerization?
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Dehydration reaction
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a polymer of less than or equal to 50 aas is called a __________. A polymer of greater than or equal to 50 aas is called a ____________.
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Peptide
Protein |
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What is the bond length of a peptide bond?
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1 & 1/2
(There are 2 resonance structures) |
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What are some characteristics of peptide bonds?
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Rigid, do not rotate
Plane established |
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Is the alpha carbon- gamma carbon bond allowed to rotate?
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Yes (only the C=N cannot rotate)
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Which conformation has a lower free energy state, trans or cis? Which is more stable?
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Trans; Trans
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In what situation would you be most likely to find cis conformation?
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Enzymes (used to provide energy for catalysis)
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Alpha carbons of two neighboring residues are usually found in the (trans/cis) conformation.
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Trans
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Preproinsulin is how many aas in length?
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110
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What are the two major factors in protein folding?
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Hydrophobicity and charge
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What does pI stand for? This is called the ____________ form.
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Isoelectric pH (the pH where all the + plus - charges equal to zero).
Zwitterion form |
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If pH is less than pI, the molecule is (+/-) charged.
If pH is greater than pI, the molecule is (+/-) charged. |
Positive
Negative |
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Which level of structure?
Local folding of the backbone |
secondary
|
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What type of bonds hold together the secondary structure?
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Hydrogen
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How are beta sheets arrangeD?
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Antiparallel pleated sheets(read N--> C)
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Where are the side groups arranged in a helix? What level of structure is the helix? What level would tell you where the side groups are located?
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Most side groups in a helix are located on the outside of the helix (when they are arrranged on the inside of the helix, it is usually a specialixed protein and the position is related to the function)
Helix- Secondary Side groups- Tertiary |
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How much is an Angstrom?
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1/10 nm
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How many residues/turn are present in an alpha-helix? What is the pitch? This forces every ___th and ___th side chain to be in contact with the 1st side chain
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n= 3.6
p= 5.4 angstroms 3rd and 4th |
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Which level of structure? The arrangement of polypeptide chains in a multichain protein.
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Quaternary
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What kinds of bonds are present in quaternary structure?
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Noncovalent (exception being the covalent bonds of disulfide bonds)
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How are the 4 subunits of Hb held together?
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Noncovalently
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Compare and contrast HbA1 and HbS
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Normal adult hemoglobin (HbA1) is a heterotetramer (a2b2). HbS has a mutation in the beta chain (position 6 Glu is replaced by Valine). This replacement of a hydrophilic aa for a hydrophobic one causes HbS to have a lower affinity for carrying O2. Also it causes deoxy-HbS to polymerize with other deoxy-HbS forming fibrils which precipitate inside the red blood cell
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What % of African Americans are homozygous for HbS? Heterozygous?
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0.4%; 10%
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What type of a sequence does a protein have that needs to be secreted?
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N-terminal signal peptide sequence
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What halts cytosolic translation for peptides that are being secreted?
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SRP (signal recognition particle)
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Describe the secretory pathway.
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1) Cytosolic ribosome synthesizes a N-terminal signal peptide sequence
2) A SRP (signal recognition particle) binds halts translation 3) The ribosome moves to the ER, where the SRP binds to a docking protein. 4) The ribosome is transferred to a translocon. Translation resumes inside the ER. 5) Protein is modified, passed to the golgi apparatus and eventually secreted from the cell |
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__- linked glycosolation is an example of cotranslational glycosylation
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N
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__- linked glycosolation is an example of posttranslational glycosylation
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O
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What sequence is necessary for N-linked glycosylation?
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Asn-X-Thr/Ser (where X is any aa except Pro or Asp)
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Explain the location of where everything happens in N-linked glycosylation
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The oligosaccharide is partially assembled on the ER cytosolic side, then flipped to the lumenal side to be linked to the amide on the Asn (after the signal peptide is already cleaved from the protein chain)
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Which type of glycosylation can affect protein folding?
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N-linked (the O-linked occurs after the protein is already folded)
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where (organell) does O-linked glycosylation occur?
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Golgi apparatus
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What sequence is required for O-linked glycosylation?
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No specific sequence is required, however an non-aromatic oxygen is required, therefore the substrate must by Ser or Thr or hydroxylated Lys
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What determines the type of sugars that are attached during glycosylation? Are the oligosaccharides heterogeneous or homogeneous?
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The types of glycosyltransferases present determine which sugars will be attached.
Heterogeneous because the glycosyltransferases compete to add the sugars. |
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What type of modifications determine where a protein goes and what is does?
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Post-translational
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Explain what happens when a protein is supposed to go to a lysosome
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Glycosyltransferases add N-acetylglucosamine phosphate (GlcNAc-P) to high mannose-type oligosaccharides on proteins destined to go to lysosomes
A glycosidase removes the GlcNAc, forming a mannose 6-phosphate which signals for that protein to be transported to the lysosomes |
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Give an example of cotranslational proteolysis
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When signal peptidase cleaves the N-terminal signal peptide sequence during entry to the ER (prior to N-linked glycosylation)
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What enzyme cleaves the connecting peptide (C-peptide) of proinsulin? What are the sites in which it is cleaved?
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carboxypeptidase E
Between T30-R31 and R65-G66 |
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Describe the structure of insulin. Can insulin be easily reformed if these disulfide bonds are broken?
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Two aa chains (A and B) held together by disulfide bonds.
NO! |
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Why does one develop an insulin resistance over time (to exogenous insulin)?
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Since bovine and porcine insulin are not excatly the same as human insulin, the body can trigger an immune response. Anti-insulin antibodies can produce an insulin resistance over time
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Which type of insulin is closest in sequence to human insulin (and therfore produces the least side effects)?
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Porcine
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What is the primary insulin used for humans?
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Recombinant
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Collagen is an example of ____________ hydroxylation and ___________ glycosylation.
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cotranslational hydroylation and posttranslational glycosylation
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Procollagen is contains cotranslational ____________ to form ____________ and ________.
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hydroxylation
3-hydroxyproline and 4-hydroxyproline |
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Where does O-linked glycosylation occur in procollagen
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On the 5-hydroxylysine (formed by cotranslational hydroxylation)
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Procollagens are ______________
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Glycoproteins
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What is the repeating sequence of procollagens?
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Gly-X-Y (where every 1/3 X positions are Pro and 1/3 Y positions are Hyp)
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What does Hyp stand for?
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3- or 4-Hydroxyproline
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The Pro or Hyp every 3 aas in procollagen forms a __________ type __ helix. What part of the chain points toward neighboring chains? What type of strong bonds does this allow for?
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Polyproline type II helix ;
carbonyl points toward neighboring chains allows for strong interchain H-bonds |
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Which type of aa in procollagen allows for an apolar edge?
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Glycine (at every 3rd residue)
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Three chains of procollagen wind around each other to form a ___________.
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Superhelix
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Where and when is procollagen cleaved to form collagen?
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At both N- and C-terminal ends once inside vesicle
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Collagen super helices assemble into fibrils by end-to-end and side-to-side aggregation. Which extracellular enzyme is responsible for cross-linking the lysine side chains to provide the collagen with its strength?
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Lysyl oxidase
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what is the final step in collagen formation?
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Cross-linking
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Cross-linking of collagen involves formation of an ___________ derivative of lysine. What is this derivative called?
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Aldehyde;
Allysine |
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Two allysines can react to form an ________________.
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Aldol cross-link
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Allysine can react with lysine to form a _______________.
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lysinonorleucine cross-link
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How many lysines (or derivatives) are needed to cross-link collagen?
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two
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Elastin uses ______ lysines to form a ____________ cross-link. This forms a rings, allowing it to stretch
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4 (3 allysines and one lysine);
desmosine. |
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What is the half life of human lens crystallines?
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NEVER replaced
|
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What is the half-life of Hb?
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60 days
|
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What is the half-life of Factor VIII?
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20 hours
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What are the 3 factors which determine protein half-life?
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1. N-terminal AA
2. Proteins rich in PEST aas are rapidly degraded 3. Tagging by Ubiquitin-protein ligase (E3) for digestion. |
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What is the half-life of Methionine?
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>20 hours
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What is the half-life of alanine?
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>20 hrs
|
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What is the half-life of Serine?
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>20 hrs
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What is the half-life of threonine?
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>20 hrs
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What is the half-life of valine?
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>20 hrs
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What is the half-life of glycine?
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>20 hrs
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What is the half-life of arginine?
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2 min
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What is the half-life of lysine?
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2 min
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what is the term for adding multiple ubiquitins to a protein?
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Polyubiquitination
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what degrades a protein which is marked by polyubiquitination?
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Proteosome
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E3 transfers activated UB to the condemned protein forming an ____________ bond to a _______.
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isopeptide; Lysine
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What happens to UBs and AAs after protein degradation by the protesome?
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They are released and recycled
|
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Apoptosis activates which enzyme which digests the cell?
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Cysteine aspartyl proteases (caspases)
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What are calpains and what do they do?
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calcium dependent thiol proteases;
perform limited proteolysis |