Term
| Describe some of the features of an α helix. |
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Definition
| The α helix is coil stabilized by intrachain hydrogen bonds between the carbonyl oxygen of a residue and the amide hydrogen of the fourth residue away. There are 3.6 amino acids per turn. The hydrogen bonds are between amino acid residues that have two intervening residues. Thus, these amino acid residues are found on the same side of the coil. The helix is almost always right-handed, although left-handed helices are, in theory, possible. |
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Term
| What does the modification involving the attachment of acetyl groups to the amino termini of a protein do? |
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Definition
| The acetylation of the amino termini of proteins is to make these proteins more resistant to degradation. |
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Term
| Why is an assay necessary for protein purification studies? |
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Definition
| An assay allows researchers to accurately measure the amount of the desired protein. This is important in determining if particular purification steps are effective in isolating the protein from the other cellular material. |
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Term
How is lactic acid dehydrogenase assayed? |
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Definition
|
It is assayed by the increase in NADH present. NADH has a unique absorbance at 340 nm, and the reaction can be monitored by the increase in absorbance at this wavelength.
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Term
How do gel-filtration and ion-exchange chromatography differ? |
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Definition
| Although both are used in purification, the properties of the column material determine how the separation is accomplished. Gel filtration is based on porous beads, and molecules are separated by size. In ion-exchange chromatography, the column material is charged with either positively or negatively charged molecules. Separation is based on the protein’s charge and affinity for the column media. |
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Term
How can a protein’s isoelectric point be used in protein purification? |
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Definition
Isoelectric focusing is an electrophoretic technique in which a gradient charge is applied. Proteins migrate through the gradient field until they reach a point at which the pH is the same as the protein’s pI. |
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Term
What is the purpose of determining the specific activity, yield, and purification level of a protein purification protocol? |
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Definition
The measurements allow one to determine if the individual steps were effective at selectively isolating the protein while maintaining its presence and activity. In order to successfully purify protein, both the yield and purification level must remain high. |
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Term
How can recombinant DNA technology aid in protein purification? |
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Definition
Large quantities of proteins can be expressed, allowing for extensive characterization of the protein. Affinity tags can be fused to the protein to aid in solid phase studies. Mature proteins that have already been modified can be generated. |
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Term
Describe the Edman degradation method for protein-sequence analysis. |
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Definition
| A pure protein is reacted with phenylisothiocyanate, which binds to the free amino terminus. Under mildly acidic conditions, the derivatized amino acid is liberated, and can be identified by chromatography. The steps are repeated to identify the next amino acid exposed at the amino terminus. |
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Term
| How can the amino acid sequences be used to design a DNA probe? |
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Definition
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Using the amino acid sequence and the genetic code, a DNA sequence can be designed. (Codon degeneracy must be considered in the design.)
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Term
| What is one advantage of using the recombinant DNA methods to determine protein sequences? |
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Definition
| Large proteins can be difficult to sequence by traditional methods because only short peptides can be sequenced. Long sections of DNA can be cloned and sequenced, and the genetic code used to determine the amino acid sequence. |
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Term
|
What is the first step in determining the structure of a protein by x-ray crystallography?
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Definition
| The first step is to prepare a protein in a crystal form, in which all protein molecules are oriented in a fixed, repeated arrangement. |
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Term
| Why are monoclonal antibodies more useful than polyclonal antibodies? |
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Definition
| Monoclonal antibodies are more specific, consisting of one type of antibody with a specific binding site and affinity. Polyclonal antibodies contain several different antibodies with slightly different binding affinities and specificities. Monoclonal antibodies are more suitable for examining protein structure and studying mechanisms. |
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Term
| Briefly describe how an ELISA works. |
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Definition
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In ELISA, the antigen of interest is complexed with a specific antibody under appropriate assay conditions, and excess antibody is removed. The antibody is complexed to an enzyme, which can be measured quantitatively using an appropriate substrate and assay tool, often by a colorimetric or radioactive product formation.
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Term
|
What are some of the advantages of NMR spectroscopy compared to x-ray crystallography?
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Definition
| While both are expensive and time-consuming techniques, NMR allows the native structure of the protein in solution to be determined. |
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Term
| In many enzyme assays, the natural substrate and product are not used. Why? |
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Definition
| Many products are difficult to measure accurately. Some are simply difficult to measure, while others are difficult to discern against the background of other molecules present in the reaction. Instead, substrates are chosen that the enzyme can still process but that result in products that can be easily measured. For example, substrates are chosen that result in products that are colored and can be detected spectrophotometrically. |
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Term
| Give an example of a reaction catalyzed by a hydrolase. |
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Definition
| Hydrolases catalyze a reaction where a water molecule is added across some linkage that was originally formed by the removal of water. Some examples are the hydrolysis of an ester, the hydrolysis of a peptide bond, the hydrolysis of a glycoside bond |
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Term
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There are six basic categories of enzymes. List the categories and define the type of reaction.
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Definition
oxidoreductases (oxidation-reduction reactions)
transferases (group transfers)
hydrolases (hydrolysis reactions)
lyases (addition or removal across a double bond)
isomerases (intramolecular group transfer)
ligases (ATP-dependent ligation of substrates). |
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Term
|
How is the substrate bound to the active site?
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Definition
| The active site is a small part of the total enzyme structure. It is usually a three-dimensional cleft or crevice, formed by amino acid residues from different regions of the polypeptide chain. The substrate is bound by multiple noncovalent attractions such as electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions. The specificity is dependent on the precise arrangement of the various functional groups in the binding site. |
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Term
| You believe a substrate fits into a cleft like a key into a lock, but your roommate does not. Who is right? |
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Definition
| You are both partially correct. Like a lock and key, the substrate fits precisely into the enzyme. However, the site is not a rigid cleft, but is flexible. Thus, it is possible for the substrate to actually modify the shape of the site a bit, a hypothesis known as induced fit. |
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Term
| In an enzymatic reaction in a test tube, the reaction will eventually reach equilibrium. Why does this not happen in living organisms? |
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Definition
| In a cell, the product may be utilized for a subsequent reaction, thus the reaction may not reach equilibrium. |
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Term
| What is the Michaelis-Menten equation? Define all parameters. |
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Definition
V0 = Vmax(S/(S + KM))
Initial velocity = V0
Maximum velocity = Vmax
Substrate concentration = S
Michaelis constant = KM
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Term
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Definition
| The maximum velocity or rate of reaction as catalyzed by a specific amount of enzyme when it is saturated with substrate. |
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Term
| What is the upper limit of kcat / KM? |
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Definition
| The diffusion-controlled interaction of the substrate and enzyme determines the upper limit of the rate. The upper limit is 108 – 109 s-1M-1. |
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Term
| How do the intermediate steps in multi-substrate enzyme mechanisms differ? |
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Definition
| In a sequential displacement reaction, both substrates bind and a ternary complex of all three is formed. In a double displacement (ping-pong), one or more products are released prior to binding of all substrates. Thus, a substituted enzyme intermediate is formed. |
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Term
| Enzyme populations are studied with the use of ensemble methods. How would the data differ from a single-enzyme study? |
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Definition
| In the ensemble method study, you would get a single value, which would represent the average of the heterogeneous assembly of all forms of the enzyme present. Single-enzyme studies enable biochemists to look into the workings of individual molecules. |
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Term
|
Explain what an abzyme is and give an example.
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Definition
| Catalytic antibodies are abzymes and are produced using transition-state analogs as antigens. An antibody made to a bent porphyrin molecule can metallate a porphyrin molecule at a rate 2500-fold faster than the uncatalyzed rate. |
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Term
| How are the types of inhibition kinetically distinguishable? |
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Definition
| Competitive inhibition can be overcome by the presence of large amounts of substrate. However, the apparent KM is increased. In noncompetitive inhibition, substrate can bind to the EI complex, however, the Vmax is decreased. In mixed inhibition, both values may be altered. |
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Term
| What is an affinity label? |
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Definition
| This is a substrate analog that is structurally similar to the substrate, binds to the active site, and chemically reacts with a residue in the active site. It is used to study enzyme structure and mechanism. |
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Term
| What are transition state analogs? |
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Definition
| These potent inhibitors mimic the structure of the transition state involved in the catalytic process. They bind very tightly to the catalytic site and are useful in determining the structure and catalytic mechanism of the enzyme. |
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Term
| What are the two systems for naming the positions of the double bonds? Provide examples. |
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Definition
| Two systems are used. One system refers to the double bond relative to the last, or omega (ω), carbon. (An example would be ω-3 fatty acids.) The other system uses notation that indicates the position of the double bond relative to the carboxyl carbon, and indicates if the bond is cis or trans. (An example would be cis-Δ9.) |
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Term
| What are some molecules that form the polar head group of a phospholipid? Provide several examples. |
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Definition
serine
ethanolamine
choline
glycerol
inositol |
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Term
| What are the primary structural differences between phospholipids constructed from a glycerol platform and those from sphingosine? |
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Definition
| Phospholipids derived from glycerol are called phosphoglycerides and consist of a glycerol backbone to which is attached two fatty-acid chains and a phosphorylated alcohol. Sphingomyelin is a phospholipid derived from sphingosine, an amino alcohol. The sphingosine backbone is linked to a fatty acid by an amide bond and the primary hydroxy group of sphingosine is esterified to phorphorylcholine |
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Term
| How are lipid bilayers formed? What is the driving force? |
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Definition
| Bimolecular sheets of lipids form spontaneously by self-assembly. Hydrophobic interactions are the driving force. Van der Waals attractive forces between the hydrocarbon tails favor the close packing of the tails. The polar heads are attracted to each other by electrostatic and hydrogen-bonding attractions. |
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Term
| Why do most phospholipids preferentially form sheets instead of micelles? |
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Definition
| The two tails in phospholipids makes it sterically unstable for them to form micelles. |
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Term
| What are liposomes? What are some of the current commercial applications? |
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Definition
| spherical structures of lipid bilayers, similar to miniature organelles. They are extremely useful as models of cell systems. They can be used to contain or transport molecules such as drugs for therapy, DNA for gene therapy, and are commonly used in cosmetics such as skin creams. |
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Term
| How is the nuclear membrane different from other membranes? |
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Definition
| The nuclear membrane is not continuous. It is a set of closed membranes that comes together at pores. |
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Term
| What is the function of prostaglandin H2 synthase-1? How does its position in the membrane facilitate its activity? |
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Definition
| Prostaglandin H2 synthase-1 converts arachidonic acid into prostaglandin H2. The protein is embedded in the membrane, with a hydrophobic channel submerged about halfway through the bilayer. The arachidonic acid is a product of membrane lipid hydrolysis and enters the protein channel from within the membrane, successfully avoiding any interaction with aqueous environments. |
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Term
| Eukaryotic cells are distinguished from prokaryotic cells by the presence of membranes inside the cell that form internal compartments. What are some examples? |
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Definition
| Peroxisomes play a major role in the oxidation of fatty acids. Mitochondria are organelles in which ATP is synthesized. The nucleus is a double membrane that consists of a set of closed membranes that come together at nuclear pores. The endoplasmic reticulum’s roles include drug detoxification and modification of proteins for secretion. |
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Term
|
What is receptor-mediated endocytosis?
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Definition
| Receptor-mediated endocytosis is a transport process in which a protein is recognized by a receptor on the cell surface and then that region of the membrane invaginates into the cell. |
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Term
| Give the net equation of the citric acid cycle. |
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Definition
Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi →
2 CO2 + 3 NADH + 3 H+ + FADH2 + GTP + CoA |
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Term
| Why is the isomerization of citrate to isocitrate a necessary step of the citric acid cycle? |
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Definition
| Citrate is a tertiary alcohol that cannot be oxidized. The isomerization converts the 3° alcohol into isocitrate, which is a 2° alcohol that can be oxidized. |
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Term
| List the five coenzymes that are required for the oxidative decarboxylation of pyruvate and α–ketoglutarate and give the essential nutrient (vitamin) that is required for each. |
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Definition
Thiamine pyrophosphate: thiamine, vitamin B1
Lipoamide: lipoic acid
NAD+: niacin
FAD: riboflavin, vitamin B2
Coenzyme A: pantothenic acid |
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Term
| Explain why a GTP is energetically equivalent to an ATP in metabolism. |
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Definition
The enzyme nucleoside diphosphokinase reversibly transfers a phosphoryl group from GTP to ADP according the reaction:
GTP + ADP ↔ GDP + ATP
Conversly, a phosphoryl group can be transferred from ATP to a GDP forming GTP. |
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Term
| Why is it necessary that there be a mechanism to replenish oxaloacetate? |
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Definition
| During periods of biosynthesis, oxaloacetate may be converted to amino acids for protein synthesis. Even if acetyl CoA levels are high, the citric acid cycle will operate at reduced levels until new oxaloacetate is formed. |
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Term
| Starting with oxaloacetate in the glyoxylate cycle, identify what molecules enter and exit the glyoxylate cycle. |
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Definition
| The cycle begins with the condensation of acetyl CoA and oxaloacetate to form citrate resulting in a net two carbon entry into the cycle. When isocitrate is hydrolyzed to succinate and glyoxylate, succinate leaves the cycle to serve as a biosynthetic intermediate in other pathways. Glyoxylate then condenses with another Acetyl CoA to form malate, allowing for another two carbon fragment to enter the cycle. |
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Term
| What is the energy source that drives the condensation of oxaloacetate and and acetyl CoA to produce citrate? |
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Definition
| Citrate synthase catalyzes the condensation of acetyl CoA and oxalacetate to form citryl CoA. This reaction is easily reversible. The hydrolysis of the thioester of citryl CoA forms citrate and regenerates the CoA. The hydrolysis of the high energy thioester drives the reaction toward citrate. |
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Term
| How does the decarboxylation of α-ketoglutatarate resemble that of pyruvate decarboxylation? |
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Definition
| Both are α-ketoacids, which are decarboxylated, and involve formation of a thioester with CoA, which has high transfer potential. The enzymatic complexes and mechanisms are similar, and the dihydrolipoyl dehydrogenase components are identical. |
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Term
| How is succinate dehydrogenase unique when compared to the other enzymes in the citric acid cycle? |
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Definition
| It is the only enzyme embedded in the mitochondrial membrane, and it is directly associated with the electron transport chain. |
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Term
| Are the acetyl carbons that enter the citric acid cycle the exact same carbons that leave as CO2? Briefly explain. |
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Definition
| No, the carbons are different. The carbons that leave as CO2 come from oxaloacetate that condensed with acetyl CoA. However, since succinate is symmetrical, and the carbons randomize, eventually all carbons are turned over. |
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Term
Membranes carry out what functions due to their electrically polarized structure? |
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Definition
| Membrane potential plays a key role in transport, energy conversion, and excitability. |
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Term
Provide a brief description of oxidative phosphorylation. |
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Definition
| It is the process in which ATP is formed, due to the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers in the inner membrane of the mitochondria. |
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Term
What is the current belief for the presence of mitochondria in eukaryotic cells? What is the proof? |
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Definition
| It is believed that the organelles are the result of an endosymbiotic event. The structure of mitochondria is consistent with this theory. Furthermore, DNA sequence analysis suggests that an ancestor of an existing bacterium is the source for extant mitochondria. |
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Term
| What is a major defense strategy against oxidative damage caused by reactive oxygen species ? |
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Definition
Superoxide dismutase converts superoxide radicals to peroxide and oxygen (requires protons), and catalase converts hydrogen peroxide to water and oxygen. |
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Term
What is the actual function of the protons in the synthesis of ATP by FoF1 ATP synthase? |
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Definition
| The proton gradient is necessary for ATP synthesis because the binding of a proton to the enzyme causes a conformational change that releases the bound ATP. The role of the proton gradient is not to form ATP but to release it from the synthase. |
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Term
| How is oxidative phosphorylation regulated? |
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Definition
| The electrons do not flow unless ADP is available to be simultaneously phosphorylated to ATP. Thus, the synthesis of ATP does not occur unless ADP levels are high. This is referred to as acceptor control. |
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Term
| What are uncouplers? Provide an example of when this might be useful. |
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Definition
| Uncouplers destroy the proton gradient across the inner membrane by carrying protons back into the matrix. This disrupts the coupling of electron transport to oxidative phosphorylation, and the energy is released as heat instead of being used to drive phosphorylation of ADP. Non-shivering thermogenesis, to generate heat for newborns, is one example provided (due to the uncoupler UCP-1). |
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Term
| Explain why carbon monoxide is toxic. |
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Definition
| Carbon monoxide bond to the ferrous ion of cytochrome a3 of cytochrome c oxidase. This blocks the electron flow to oxygen and the proton-motive force can no longer be generated. Without the proton gradient, the phosphorylation of ADP cannot occur. Thus, energy production ceases. |
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Term
| Briefly outline the steps in fatty acid degradation. |
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Definition
An activated acyl group is oxidized by FAD, hydrated, oxidized by NAD+ to form a second carbonyl, and then cleaved by thiolysis. |
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Term
| In odd-numbered fatty acid chains, how is the final 3-carbon piece, propionyl CoA, utilized? |
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Definition
| It is carboxylated to form D-methylmalonyl-CoA, isomerized to L-methylmalonyl-CoA, and then converted to succinyl CoA, which enters the citric acid cycle. |
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Term
| What is the role of the acyl carrier protein in fatty acid synthesis? |
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Definition
| Intermediates in fatty acid synthesis are covalently linked to the sulfhydryl groups of the ACP. The energy for the condensation reactions comes from the hydrolysis of these high energy thioester bonds. |
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Term
| Explain why animals are unable to convert fatty acids to glucose? |
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Definition
| The synthesis of glucose via gluconeogenesis requires pyruvate or oxaloacetate. In animals, the only metabolic route to convert acetyl-CoA to oxaloacetate is through the citric acid cycle, which involves the loss of two CO2. So animals cannot bring about the net conversion acetyl CoA into pyruvate or oxaloacetate. Plants and bacteria possess the glyoxylate cycle, which can convert two acetyl groups into succinate. |
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Term
| What is the fate of glycerol released during lipolysis? |
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Definition
| The glycerol is absorbed by the liver, phosphorylated, and then oxidized to DHAP, which is isomerized to glyceraldehyde 3-phosphate. It can then become either glucose or pyruvate. |
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Term
| How do taste and odor detection vary? |
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Definition
| Each neuron detecting odor expresses one unique receptor, and the receptor transmission of the common receptors converge to specific sites. In contrast, neurons for taste sensation each express several different tastant receptors, which are transmitted to the brain. |
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Term
| What evidence is there that sweet receptors are heterodimeric? |
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Definition
| Studies in knockout mice indicate that two memebers of a second 7TM family (T1R1 and T1R3) are expressed simultaneously. Individually, the two respond only weakly to sweet testants. |
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Term
Describe the steps leading to detection of light. |
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Definition
| A photon of light causes a conformational change in 11-cis-retinal, deprotonating the Schiff base and inducing a conformational change in the rhodopsin. This transmembrane protein activates a G protein, transducin. Transducin is altered by GTP/GDP exchange, leading to activation of a phosphodiesterase, which converts cGMP to GMP. The decrease in cGMP causes gated ion channels to close, and the membrane becomes hyperpolarized causing neuronal signaling. |
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Term
|
What is the reason for color-blindness in humans?
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Definition
| The genes for the visual pigments are very similar, some as much as 98% identical. During replication, these regions undergo unequal homologous recombination. This can occur between or within genes, resulting in loss, gain, or formation of hybrid genes. |
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Term
|
How is sound converted to a biochemical signal?
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Definition
| As sound passes across the hair bundles in the stereocilia, a slight deflection occurs, which tips the bundle. This tip, depending on its direction, causes ion channels to open or close, altering the potential of the membrane. |
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Term
| How are the molecular mechanisms for salty taste sensations and non-painful touch sensations similar? |
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Definition
| Both are a combination of sensory systems expressed in a common organ. Amiloride-sensitive Na+ channels appear to play a role. |
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Term
| Name another stimulus (besides the basic senses) that appears to be detected by humans? |
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Definition
| Humans can also detect pheromones, and our circadian rhythms are influenced by daily changes in light exposure. |
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Term
| Many of the receptors involved in transmission of stimuli contain multiple transmembrane domains. Why? |
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Definition
| Several parts of the protein, the transmembrane regions, assemble together in order to form the channel or pore. A single a helix or b sheet cannot form a pore large enough to allow ions to pass. |
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Term
| How is the immune system diversity similar to evolutionary processes? |
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Definition
In evolution, there is a mechanism to promote diversity, and then specific traits are selected for or against. This is also true of the immune system. The genes for antibodies and T-cell receptors are the product of genetic shuffling and recombination. Selection processes select against those cells that synthesize dangerous antibodies and for those that can aid in the immune response. |
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Term
| What are the two lines of defense in our immune system? |
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Definition
| The innate immune system responds rapidly to specific features present in most pathogens. The adaptive immune system responds to specific features present only in a given pathogen. |
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Term
| What are Toll-like receptors and what pathogens do they recognize? |
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Definition
| Toll-like receptors (TLRs) are receptors of the innate immune system containing leucine-rich repeats. These receptors are expressed in cell membranes for the detection of extracellular pathogens such as fungi and bacteria. |
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Term
| What types of bonds are present in antigen:antibody binding? |
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Definition
| The binding involves the apposition of shapes that are complementary. The bonds include van der Waals contacts, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. |
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Term
| Explain why the amino terminal immunoglobulin domains of the L and H chains have hypervariable sequences. |
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Definition
| Each domain contains three loops that, when the L and H chains come together, form six loops at the end of each arm. These ends are the antigen binding site, and a very large number of different binding sites can be constructed by combinatorial association. |
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Term
How are pathogens inside cells destroyed by the immune system? |
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Definition
| The cells are destroyed by a mechanism that flags the presence of pathogens inside the cell. Protease digests proteins inside the cytosol, such as those harbored in a virus. The proteins are joined to MHC proteins in the ER and then displayed on the cell’s surface. When recognized by T cells as foreign particles, a series of steps induces apotosis of the cell. |
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Term
What are the steps in T-cell activation? |
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Definition
| T-cell receptors bind the MHC peptide complex, and the CD8 interaction with the MHC allows kinase phosphorylation of the CD3 complex. This generates attachment sites for ZAP-70, which can phosphorylate other targets in the signaling cascade. Other proteins (CD45 and CD28) also participate. |
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Term
| How does the response differ when a peptide that binds to a class II MHC is compared to one that binds to a class I MHC? |
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Definition
Binding to class II MHC is a signal that a pathogen has been detected, whereas binding to class I MHC indicates a cell has succumbed and signals for cell destruction. |
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Term
| Why is it difficult to develop a vaccine for AIDS? |
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Definition
| The HIV strains have very inefficient replication, resulting in gene mutations that result in variations in coat proteins. |
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