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Flashcards in Insulin Deck (40)
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1

physiological activities of insulin

stimulates glucose and amino acid transport (hepatocytes, muscle cells)
increase glycogen synthase activity
increase protein synthesis and decrease protein degradation
depresses lipolysus

2

insulin protein structure

B chain
A chain
C peptide

3

what is needed to form dimers

hydrophobic interactions at c terminus of b-chain critical to form dimers

4

what can zinc be associated with

3 insulin monomer at hisB10 residue of each monomer

5

what is the purpose of phenolic species

bind to specific sites on insulin hexamers causing conformational change that increases the chemical stability of insulin in commercial preparations

6

what is the T-R transition

shifting the conformational equilibrium from an extended structure (tstate) to and alpha helical structure (rstate)
strengthens association of insulin molecule

7

describe the association of insulin

starts as a monomer --- dimer ---- higher order associated states----- zinc forms hexamer (T6) ------ phenolic preservative froms hexamer (R6)

8

what is an insulin analog

modifications where changes are made in the amino acid sequence of the insulin molecule that affect the duration of action

9

how to you achieve site directed mutagenesis

determine site you want to mutate
clone gene cDNA into a selectable plasmid
amplify sequence by PCT
remove unamplified gene sequence
identify success of site mutageneiss

10

ways to identify success of site mutagenesis

transform into bacteria
antibiotic selection of positive clone
restriction enzyme digestion and sequencing gene
compare with normal gnee sequence

11

melting temp of site directed mutagenesis primers

78 or greater

12

3'end of site directed mutagenesis primer should end on

C or G

13

where should the mutation be on site directed mutagenesis primers

in the middle of the primer

14

site directed mutagenesis primers should be how long and how much GC content

25-45 nucleotides long
GC content of 40% at least

15

steps to remove unamplified DNA sequence

methylate plasmid
demature the plasmid
anneal(hot then cold) the oligonucleotide primers containing the desired mutation
use pfuturbo polymerase extend and incorporate the mitagenic primers
results in nicked circular strands
digest the methylated non mutated parental dna templat with dpn I
transform the circular nicked dsDNA into supercompetent cells

16

how can you identify a mutated sequence

transform into bacteria
grow a colony
isolate plasmid dna from bacteria
OR
digest with restriction enzymes BamH1 and XHo1 and send to sequencing facility
OR compare with original

17

changes on humalog lispro and the result

lysine and proline at the end of the bchain are reversed
results in greater steric inderence so a reduced ability to form insulin dimers and hexamers. but does not alter receptor binding
allows larger amounts of active monomeric insulin to be immediately available

18

novorapid aspart changes and result

proline amino acid at the end of the bchain is changed to aspartic acid
results in greater charge repulsion and steric hindrance to be absorbed quickly into the bloodstream

19

glargine is synthesized from

ecoli

20

glargine: two positively charged arginine to cterminus of the b chain - whats the result

shifts isoelectric point from 5.4-6.4

21

glargine: asparagine at position 21 in the a chain is replaced by glycine, results in

prevents deamination (loss of amino group) and dimerization (production of polymers) of arginine residue

22

what is the purpose of shifting the isoelectric point to 6.7 in glargine

makes the insulin less soluble at physiological pH and more soluble in acidic
at the injection site the pH is 4 so the acidic insulin solution precipitates
the precipitate slowly dissolves causing gradual release of monomers into the blood

23

ways to delay the release of insulin in glargine

shift isoelectric point to pH 6.7
add zinc

24

insulin detemir synthesized from

bakers yeast

25

modifications to detemir

14 carbon FA chain attached to the lysine residue at position 29 of insulin B chain
remove threonine from position 30 of the insulin B chain

26

purpose of fatty acid chain in detemir

allos insulin to be formulated as a neutral solution that doesnt precipitate after injection
contributes to hexamer formation and delays hexamer dissociated allowing the solubilized insulin to remain in depot
allows insulin to be 99% albumin bound buffering aginst sudden changes in insulin concentration and absorption thus reducing risk of hypoglycemia and albumin binding also acts to slow diffusion of insulin into the interstitial compartment

27

why does lente have intermediate action

due to crystallization and zinc hexamer formation which increases duration of action and slows onset time

28

what is protamine and its use in insulin

small proteins composed greatly of arginine
when mixed with insulin slows down onset and extends duration of action

29

describe the physical properties of NPH insulin

small rod shaped crystals
allows extended time action because of the required dissolution time

30

how is the NPH insulin prepared

by cocrystallization of zinc hexamers of insulin with protamine