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Year 3 - Biotechnology > 5 - Insulin > Flashcards

5 - Insulin Flashcards

1
Q

Blood glucose regulation

A
  • High BG stimulates insulin release; low BG stimulates glucagon release
  • Glucagon stimulates glycogen breakdown
  • Insulin stimulates glycogen formation and stimulates glucose uptake from blood
  • Glycogen broken down into glucose (and glucose formed into glycogen) in the liver
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2
Q

Physiological activities of insulin

A
  • Stimulates glucose transport (muscle cells, lipocytes, hepatocytes)
  • Stimulates amino acid transport (hepatocytes, muscle cells)
  • Increases glycogen synthase activity (hepatocytes)
  • Increases protein synthesis and decrease protein degradation (hepatocytes, muscle cells)
  • Depresses lipolysis (lipocytes)
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3
Q

Insulin structure

A

Signal peptide – B-chain – C-peptide – A-chain

  • Signal peptide = 24 aa
  • Peptide (B chain) = 30 aa
  • Propeptide (C peptide) = 31 aa
  • Peptide (A chain) = 21
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4
Q

Function of C-peptide of insulin

A

To measure insulin levels in the blood

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5
Q

Intrinsic property of insulin (charge)

A
  • Net charge of the molecule
    • Negatively charged at neutral pH
    • Net charge on insulin molecule produced from the ionization potential of 4 glutamate residues (found in A and B chains), 2 histidine residues (found in B chain), lysine residue (found in B chain), arginine residues (found in B chain), and 2 alpha-carboxyl and 2 alpha-amino groups
    • Insulin has isoelectric point (pI) of 5.3 in the denatured state
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6
Q

Different association force of insulin molecule

A
  • Hydrophobic interactions at the C-terminus of the B-chain are critical for formation of dimers
  • Zinc molecules can be associated w/ 3 insulin monomers at HisB10 residue of each monomer
  • Phenolic species (ex: phenol, m-cresol, or methylparaben) bind to specific sites on insulin hexamers, causing a conformational change that increases the chemical stability of insulin in commercial preparations
    • Phenolic ligands are bound w/ insulin by H-bonds w/ the carbonyl oxygen of CysA6 and the amide proton of CysA11 as well as numerous van der Waals contacts in a binding pocket between monomers of adjacent dimers
    • Binding of these ligands stabilizes a conformation change that occurs at the N-terminus of the B-chain in each insulin monomer
  • Shifting the conformational equilibrium of residues B1 to B8 from an extended structure (T-state) to an alpha-helical structure (R-state) strengthens association of insulin molecule
    • This conformational change is referred to as the T-R transition
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7
Q

Association of insulin

A

Monomer dimer higher-order associated states (Zn2+) hexamer (T6) (phenolic preservative) hexamer (R6)
**Monomer is the simplest form that is used in the cells; dimer doesn’t work in the cell

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8
Q

What are insulin analogs?

A

Modifications of natural insulin, where changes are made in the AA sequence of the insulin molecule that affect the duration of action

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9
Q

Describe the amino acid substitutions at A21, B28 and B29 for humulin and novolin

A
  • A21 = Asn
  • B28 = Pro
  • B29 = Lys
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10
Q

Describe the amino acid substitutions at A21, B28, B29, and B30 for porcine and bovine insulin

A
  • A21 = Asn
  • B28 = Pro
  • B29 = Lys
  • B30 = Ala (everything else = Thr)
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11
Q

Describe the amino acid substitutions at A21, B28 and B29 for lispro

A
  • A21 = Asn
  • B28 = Lys
  • B29 = Pro
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12
Q

Describe the amino acid substitutions at A21, B28 and B29 for aspart

A
  • A21 = Asn
  • B28 = Asp
  • B29 = Lys
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13
Q

Describe the amino acid substitutions at A21, B28, B29, B31, and B32 for glargine

A
  • A21 = Gly
  • B28 = Pro
  • B29 = Lys
  • B31 = Arg
  • B32 = Arg
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14
Q

Describe the amino acid substitutions at A21, B28 and B29 for detemir

A
  • A21 = Asn
  • B28 = Lys-(N-tetradecanoyl)
  • B29 = Pro
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15
Q

Steps of site-directed mutagenesis of insulin

A
  • Determine which site you want to mutate
  • Clone gene cDNA into a selectable plasmid
  • Amplify gene sequence by PCR
  • Remove un-amplified gene sequence
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16
Q

Sites to mutate on regular insulin to make rapid acting lispro

A
  • Proline replaced w/ lysine at B28

- Lysine replaced w/ proline at B29

17
Q

Design of site-directed mutagenesis primers

A
  • Mutation should be in the middle of the primer
  • Primers should be 25-45 nucleotides long and have a GC content of at least 40%
  • Melting temp should be 78 C or greater
  • 3’-end of the primer is better to end on a C or G
18
Q

PCR to remove un-amplified DNA sequence

A
  • Methylate plasmid first
  • Denature the plasmid and anneal the oligonucleotide primers containing the desired mutation
  • Using the non-strand-displacing action of PfuTurbo polymerase, extend and incorporate the mutagenic primers resulting in nicked circular strands
  • Digest the methylated, nonmutated parenteral DNA template w/ Dpn 1
  • Transform the circular, nicked dsDNA into super-competent cells
19
Q

Identification of mutated sequence

A
  • Transform into bacteria
  • Pick up a colony
  • Isolate plasmid DNA from bacteria
  • Digest w/ restriction enzymes (BamH1 and Xho1)
  • Send out to sequencing facility
  • Compare DNA sequence w/ original sequence
20
Q

Describe the dissociation of regular insulin after SQ injection

A

Hexamer (R6) hexamer (T6) dimers monomers

  • Only monomer can bind to receptor
  • Dimer and monomer can cross biological membrane and interact w/ cells
  • Phenolic preservative heavily concentrated around hexamer (R6) but gradually released as insulin dissociates into monomers
21
Q

Lispro (Humalog) – describe the changes made to the structure and what impact they have

A
  • Lysine and proline at the end of the B-chain are reversed => greater steric hindrance and reduced ability to form insulin dimers and hexamers
  • Doesn’t alter receptor binding
  • Allows larger amounts of active monomeric insulin to be immediately available for postprandial injections
22
Q

Aspart (NovoRapid) – describe the change made to the structure and what impact it has

A
  • Proline at the end of the B-chain is changed to aspartic acid => greater charge repulsion and steric hindrance due to local conformational change in the B-chain
  • To be absorbed quickly into the bloodstream
23
Q

Glargine (Lantus) – describe the changes made to the structure and what impact they have

A
  • Synthesized from non-disease producing strain of E. coli
  • Modification of 3 amino acids:
    • 2 positively charged arginine molecules added to the c-terminus of the B-chain -> shifts pI from 5.4 to 6.4
    • Asparagine at position 21 in the A chain is replaced by glycine -> prevents deamination (loss of amino group) and dimerization (production of polymers) of the arginine residue
  • The shift in isoelectric point from 5.4 to 6.7 makes insulin less soluble at physiological pH (7.4) and more soluble at acidic pH
  • Glargine formulated at pH 4
    • At injection site (pH 7.4), the increase in pH causes acidic insulin solution to precipitate
    • Precipitate slowly dissolves, causing gradual release of monomers into blood
  • Release of insulin further delayed by addition of Zn2+ to the formulation
24
Q

Detemir (Levemir) – describe the changes made to the structure and what impact they have

A
  • Synthesized from bakers’ yeast
  • Modifications:
    • 14 carbon fatty acid chain (myristic fatty acid or tetradecanoil) is attached to the lysine residue on position 29 of insulin B-chain -> promotes self-association of insulin molecules and binding to albumin at injection site
    • Removal of threonine from position 30 of the insulin B-chain
  • Fatty acid chain:
    • Allows insulin to be formulated as a neutral solution which doesn’t precipitate after injection
    • Contributes to hexamer formation and delays hexamer dissociation allowing the solubilized insulin to remain in depot
    • Allows insulin to be 99% albumin-bound, which buffers against sudden changes in insulin concentration and absorption thus reducing risk of hypoglycemia
  • Albumin binding also acts to slow diffusion of insulin into interstitial compartment
25
Q

Summary of insulin structure

A
  • 2 zinc molecules help insulin to form hexamer
  • Extra zinc molecules on outside of insulin hexamer increase association of insulin molecule
  • Phenolic species help to induce T -> R transition
  • pH shift from neutral to acidic helps insulin to precipitate at physiological pH
  • Addition of fatty acid allows insulin binding to albumin
  • Addition of a protein (ex: protamine) helps form an insulin-protamine complex
  • Formation of crystal delays release of insulin
26
Q

Intermediate acting insulin – examples and what general changes are made

A
  • NPH (neutral protamine Hagedorn) – insulin co-crystallized w/ protamine; antigenic properties
  • Lente – insulin formulated w/ zinc; intermediate action due to crystallization and zinc hexamer formation which increases duration of action and slows onset time
27
Q

NPH insulin – describe the changes made to the structure and what impact they have

A
  • Neutral crystalline suspension prepared by co-crystallization of zinc hexamers of insulin w/ protamine
  • Protamine = small proteins composed greatly of arginine; when mixed w/ insulin, slows down onset and extends duration of action
  • Very minimal levels of soluble insulin or protamine in solution
  • Crystalline formulation of insulin allows an extended time to action b/c of required dissolution time
28
Q

Lente insulin – describe the changes made to the structure and what impact they have

A
  • Preparation of 2 insoluble insulins w/ zinc (70% rhombohedral zinc insulin crystals, 30% amorphous insulin particles)
  • Forms hexamers (6 insulin molecules, 2 zinc atoms, 6 water molecules)
  • Excess zinc binds to outside of hexamers, increasing dissolution time
  • Only monomers are physiologically active, dissociation is required => extended time-action effect
29
Q

Ultralente insulin – describe the changes made to the structure and what impact they have

A
  • Like NPH, is a crystalline insulin formulation
  • Larger rhombohedral crystals
  • No protamine
  • Crystallized at pH 5.5 w/ zinc, NaCl and acetate buffer
  • Adjusted to pH 7.4 and addition of excess zinc and methylparaben as a preservative
30
Q

Premixed insulin

A
  • Several different mixes available, the following are available in Canada:
    1. Humalog mix 25 = 25% lispro (rapid acting) and 75% lispro protamine (intermediate acting)
    2. Humulin (20/80, 30/70) = 20% regular insulin and 80% NPH insulin (intermediate acting)
    3. Novolin ge (30/70, 40/60, 50/50) = 30% Toronto (regular insulin) and 70% NPH insulin (intermediate acting)
31
Q

Chemical stability of insulin formulations

A
  • Hydrolytic transformation of amide to acid groups
    • Asparagine (AsnA21) is the primary degradation mechanism of insulin formulation at acidic pH (asparagine -> aspartic acid)
    • Deamidation of AsnB3 is the primary degradation mechanism insulin formulation at neutral pH (asparagine -> aspartic acid or iso-aspartic acid)
  • Formation of covalent dimers and higher order polymers
    • High molecular weight protein (HMWP) can be formed at both storage and room temp; higher temps = higher order insulin oligomers
    • Rate of HMWP formation can be affected by strength of insulin formulation and addition of glycerol as an isotonicity agent
    • Biopotency of HMWP is significantly less (1/10 to 1/5 of insulin) than monomeric species
32
Q

Physical stability of insulin formulations

A
  • Mediated by non-covalent aggregation of insulin
  • Hydrophobic forces typically drive the aggregation although electrostatics plays a subtle but important role
  • Aggregation leads to a loss in potency of the formulation
  • Physical changes in soluble formulations = colour or clarity change, formation of precipitate
33
Q

Amino acid modifications of insulin

A
    • Disulfide bond 31 -> 96 interchain
    • Disulfide bond 43 -> 109 interchain
    • Disulfide bond 95 -> 100
34
Q

How do you identify success of site mutagenesis of insulin?

A
  • Transform into bacteria
  • Antibiotic selection of positive clone
  • Restriction enzyme digestion and sequencing gene
  • Compare gene sequence w/ normal gene sequence
35
Q

Which codons encode lysine?

A

AAA and AAG

36
Q

Which codons encode proline?

A

CCU, CCC, CCA, and CCG

Year 3 - Biotechnology (11 decks)

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