Kaplan Chapter 3 Nonenzymatic Protein Function and Protein Analysis Flashcards

Question

What is SDS-PAGE?

Answer 1

SDS-PAGE denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel.

Answer 2

Isoelectric focusing separates proteins by their isoelectric point (pI); the protein migrates toward an electrode until it reaches a region of the gel where pH = pI of the protein.

Answer 3

Protein structure is primarily determined through X-ray crystallography aer the protein is isolated, although NMR can also be used.

Answer 4

Amino acid composition can be determined by simple hydrolysis, but amino acid sequencing requires sequential degradation, such as the Edman degradation. [The Edman degradation selectively and sequentially removes the N-terminal amino acid of the protein, which can be analyzed via mass spectroscopy.]

Answer 5

Activity levels for enzymatic samples are determined by following the process of a known reaction, often accompanied by a color change.

Answer 6

Protein concentration is also determined colorimetrically, either byUV spectroscopy or through a color change reaction. BCA assay, Lowry reagent assay, and Bradford protein assay each test for protein and have different advantages and disadvantages. The Bradford protein assay, which uses a color change from browngreen to blue, is most common

Answer 7

Cytoskeletal proteins tend to be fibrous with repeating domains, while motor proteins tend to have ATPase activity and binding heads. Both types of protein function in cellular motility.

Answer 8

False. An enzyme is a protein or RNA molecule with catalytic activity, which motor proteins do have. Motor function is generally considered nonenzymatic, but the ATPase functionality of motor proteins indicates that these molecules do have catalytic activity

Answer 9

If the binding protein is present in sufficiently high quantities relative to the substrate, nearly all substrate will be bound despite a low affinity.

Answer 10

Cadherin - Two cells of the same or similar type using calcium Integrin - One cell to proteins in the extracellular matrix Selectin - One cell to carbohydrates, usually on the surface of other cells

Answer 11

Antigen–antibody interactions can result in neutralization of the pathogen or toxin, opsonization (marking) of the antigen for destruction, or creation of insoluble antigen–antibody complexes that can be phagocytized and digested by macrophages (agglutination).

Answer 12

Ungated channels are always open.

Answer 13

Transport kinetics display both Km and vmax values. They also can be cooperative, like some binding proteins. However, transporters do not have analogous Keq values for reactions because there is no catalysis.

Answer 14

Isoelectric focusing and ion-exchange chromatography both separate proteins based on charge; the charge of a protein in any given environment is determined by its isoelectric point (pI).

Answer 15

Native PAGE allows a complete protein to be recovered after analysis; it also more accurately determines the relative globular size of proteins. SDS-PAGE can be used to eliminate conflation from mass-to-charge ratios.

Answer 16

The protein of interest may not elute from the column because its affinity is too high or it may be permanently bound to the free receptor in the eluent.

Answer 17

True. The small pores in size-exclusion chromatography trap smaller particles, retaining them in the column.

Answer 18

Protein isolation is generally only the first step in an analysis. The protein identity must be confirmed by amino acid analysis or activity. With unknown proteins, classification of their features is generally desired.

Answer 19

Contamination of the sample with detergent or SDS could yield an artificially increased protein level, leading to lower activity than expected (because the protein concentration was calculated as higher than its actual value). Alternatively, the enzyme could have been denatured during isolation and analysis.

Answer 20

False. The Edman degradation proceeds from the amino (N-) terminus.

Answer 21

B. Sodium dodecyl sulfate is a detergent and will digest proteins to form micelles with uniform negative charges. Because the protein is sequestered within the micelle, other factors such as charge of the protein and shape have minimal roles during separation. In essence, the protein micelles can be modeled as being spheres, dependent only on size.

Answer 22

D From the given choices, all of them are involved in cell movement with the exception of (D). Centrioles are composed of microtubules, but are involved in mitosis, not cell migration.

Answer 23

C The most prevalent extracellular proteins are keratin, elastin, and collagen. Tubulin and actin are the primary cytoskeletal proteins, while myosin is a motor protein.

Answer 24

C For a ligand present in low quantities to have a strong action, we expect it to initiate a second messenger cascade system. Second messenger systems amplify signals because enzymes can catalyze a reaction more than once while they are active, and often activate other enzymes. Both enzyme-linked receptors and G protein-coupled receptors use second messenger systems, while ion channels do not.

Answer 25

D Ions that are not readily accessible in the cytoplasm or extracellular space are likely to be bound to a binding protein. Classically, calcium and magnesium are protein-bound. Without this background knowledge, the question can still be answered. Sodium, (A), and potassium, (B), must exist in their free states to participate in action potentials. Chloride, (C), is readily excreted by the kidney, which would not be true if it were protein-bound. Calcium must be sequestered in both the bloodstream and intracellularly because calcium is used for muscle contraction, exocytosis (of neurotransmitters and other signals), and many other cellular processes that must be tightly regulated.

Answer 26

A Antibodies are specific to a single antigen. Each B-cell produces a single type of antibody with a constant region that is specific to the host and a variable region that is specific to an antigen.

Answer 27

A The resting membrane potential is displayed by cells that are not actively involved in signal transduction. Ungated or “leak” channels permit limited free flow of ions, while the sodium–potassium pump is also active and corrects for this leakage. Ligand-gated and voltage-gated channels are involved in cell signaling and in the pacemaker potentials of certain cells, but cause deviation from—not maintenance of—the resting membrane potential.

Answer 28

D All trimeric G proteins have α, β, and γ subunits—(A), (B), and (C), respectively. Gs , Gi , and Gq are subtypes of the Gα subunit of the trimeric G protein and differ depending on the G protein-coupled receptor’s function.

Answer 29

B The proteins described in the question differ primarily in their molecular weights. eir pI values are very close, so ion-exchange chromatography, (A), is not a good choice. e question specifies a large quantity, which is better processed through chromatography than through electrophoresis— (C) and (D)—because the gel can only handle a small volume of protein.

Answer 30

C The overall pI of a protein is determined by the relative number of acidic and basic amino acids. e basic amino acids are arginine, lysine, and histidine, and the acidic amino acids are aspartic acid and glutamic acid. Glycine’s side chain is a hydrogen atom, so it will have the least contribution of all the amino acids.

Answer 31

C The gel in isoelectric focusing uses a pH gradient. When a protein is in a region with a pH above its pI, it is negatively charged and moves toward the anode. When it is in a pH region below its pI, it is positively charged and moves toward the cathode. When the pH equals the pI, the migration of the protein is halted

Answer 32

C UV spectroscopy is best used with conjugated systems of double bonds. While the double bond in the peptide bond does display resonance, this is not adequate for UV absorption. However, aromatic systems are conjugated, and phenylalanine, tyrosine, and tryptophan all contain aromatic ring structures

Answer 33

B Protein activity and concentration are generally correlated. Because we have a high concentration of protein, we expect a high activity unless the protein has been damaged or inactivated in some way. e protein could have been inactivated by experimental conditions like detergents, heat, or pH; however, these are not answer choices. Rather, we must consider how the experimental procedure works. Protein elutes off of an affinity column by binding free ligand. In this situation, the binding may not have been reversed and thus the free ligand competes for the active site of the enzyme, lowering its activity

Answer 34

A The selective cleavage of proteins by digestive enzymes allows fragments of different lengths with known amino acid endpoints to be created. By cleaving the protein with several different enzymes, a basic outline of the amino acid sequence can be created.

Answer 35

Blotting is a method of transferring fragments of protein, DNA, or RNA that have been separated via electrophoresis and applying them to a blotting membrane You can then visualize the fragments with a colored stain

Answer 36

In isoelectric focusing, the gel has a pH gradient and the proteins will migrate through the gel until they reach the pH that matches their isoelectric point At the pI, the protein has a neutral charge, so it will no longer be attracted to the anode or the cathode and it will stop migrating

Answer 37

Selectins allow cells to adhere to carbohydrates on the surfaces of other cells and are most commonly used in the immune system

Answer 38

Integrins have two membrane-spanning chains and permit cells to adhere to proteins in the extracellular matrix

Answer 39

Enzyme-linked receptors participate in cell signaling through extracellular ligand binding and initiation of 2nd messenger cascades

Answer 40

Voltage-gated channels open within a range of membrane potentials

Answer 41

In gel electrophoresis: Small molecules move fast Large molecules move slow Shorter molecules migrate more easily through the pores of the gel

Answer 42

Ligand-gated channels open in the presence of a specific binding substance, usually a hormone or neurotransmitter

Answer 43

In SDS-PAGE, you can add an additional reducing agent called 2-mercaptoethanol to reduce disulfide bonds. This further denatures the protein

Answer 44

In gel electrophoresis: Agarose gel has large pores so it is used to separate nucleic acids Polyacrylamide gel has small pores so it is better for separating proteins Nucleic acids are usually larger than proteins

Answer 45

Agrose: Horizontal, Bigger gaps in Gel, seperates both large and small but better for larger molecules, CANNOT tell the difference between sizes of bands, Mostly DNA Polyacrylamide: Vertical, smaller gaps in gel, seperates both large and small but better for smaller molecules, CAN tell the difference between sizes of bands, Mostly DNA or proteins

Answer 46

Ion channels are protein molecules that span across the cell membrane allowing the passage of ions from one side of the membrane to the other Examples of ion channels include: Ungated channels (always open) mechanically-gated channels voltage-gated channels ligand-gated channels

Answer 47

Western blotting is used to detect a specific protein in a sample

Answer 48

Gel electrophoresis is a technique commonly used in laboratories to separate charged molecules like DNA, RNA and proteins according to their size and charge Small molecules move fast Large molecules move slow

Answer 49

Native-PAGE is a polyacrylamide gel electrophoresis method for proteins using non-denaturing conditions It is run in the absence of sodium dodecyl sulfate (SDS), which would denature the proteins

Answer 50

SDS-PAGE denatures proteins and separates them solely based on mass This allows you to estimate the protein’s molecular mass

Answer 51

G protein-coupled receptors detect molecules outside the cell then activate internal signal transduction pathways and, ultimately, cellular responses The 1st messenger ligand initiates the 2nd messenger and the cascade response

Answer 52

Sodium dodecyl sulfate (SDS) denatures proteins and gives them a uniform negative charge

Answer 53

Ungated ion channels are always open