115 Flashcards
2 major functions of HSA
- as a transporter of smaller, mostly hydrophobic molecules
- as the major contributor (80%) of the osmotic swelling pressure of blood plasma
where does albumin rapidly migrate
positive electrode
forest plot
A forest plot is often used in a meta-analysis of the effect of perhaps a drug or a treatment. This type of analysis takes lots of studies which have examined the same thing, and shows the outcome of each, and of them all added together.
a-domain structure
commonly in proteins its found in isolated a-helices but this isn’t that stable so a-helices tend to be packed pairwise in proteins with their hydrophobic residues pointing towards the molecules core
HSA Mr
66 500
HSA pl
5.67
HSA structure
single polypeptide chain
585 amino acids with 17 intra-chain disulphide bonds
These disulfide bonds pull the molecule into a series of large (L) and small (S) double loops.
Not only are there three similar domains but examination of the amino acid sequence shows that there are repetitions even from one individual loop to another.
The AA sequence of albumin has an unusually high percentage of Cys (35 out of 585 amino acids).
true or false
true
in circulating plasma how much of free sulfhydryl Cys-34 is oxidised
30%
does albumin contain multiple binding sites
yes - notable for long-chain fatty acids, small heterocyclic or aromatic carboxylic acids and for metals
what ions can cys-34 (SH-group) bind
Cd, Au, Hg, Ag
what does N-terminal His-3 bind in albumin acting as a transporter
Cu(II), Ni (II)
examples of drugs that are transported by albumin
aspirin
AZT
penicillin and Warfarin
why is drugs transported by albumin pharmacologically important
This is pharmacologically important as drugs compete for binding sites and small molecules, not transported on proteins, may be excreted by the kidney into the urine.
what does serum albumin (1e7a) bind to
propofol (an anaesthetic)
what does serum albumin (1e7b) bind to
halothane (an anaesthetic)
does evans blue have a high affinity for serum albumin
yes
serum albumin excitation peaks - evans blue
peaks at 470nm and 540nm and an emission peak at 680nm
what is evans blue used as
viability assay
permeability of the blood-brain barrier to macromolecules
which of the following are albumin functions
structural
catalytic
second messenger
transport
oncotic regulator
transport
oncotic regulator
human serum albumin is rich in what
a-helices
disulphide bonds
can albumin transport different substances in several sites of its structure
yes
what organ is responsible for synthesising HSA
liver
the EF hand overview
associated with ca+ binding
named from the Ca2+ binding site between the E and F a-helices in parvalbumin.
A HELIX-LOOP-HELIX configuration characterises the EF hand
whats a motif
a conserved amino acid sequence alignment- a local alignment corresponding to a region whose function or structure is known, or its significance may be unknown
Kd definitition
it is a measure of the affinity between 2 molecuels
The Kd is the concentration at which 50% of the ligand is free and 50% is bound to a receptor.
if the Kd is low then the affinity of the ligand for the receptor is very what ?
high
if the Kd is high then the affinity of the ligand for the recepter is what ?
very low
at ligand conc above the Kd where is most the ligand
bound to the receptor
at the ligand conc below the Kd where is most of the ligand
its free
where are members of the superfamily of EF-hand containing proteins found
in the cytosol
what affinity does EF-hand containing proteins bind calcium
Kd of around 10^-6
extracellular [Ca+] M
10^-3
intracellular (unstimulated) [Ca+] M
10^-7
intracellular (stimulated) [Ca+] M
10^-5/ 10^-6
how many residues does EF-hand contain
29 resdidues
what amino acid is at position one in the first a-helix of the EF-hand
Glu
positions of hydrophobic residues facing the core of the molecules (first a-helix)
2,5,6,9
position of glu and residues on the second a-helix of EF-hand
glu at psotion 21
residues at 22,25,26,29 and they are hydrophobic
Acidic amino acids have carboxylate oxygen atoms that can ligate calcium.
true or false
true
Glycine found at position 15 permits a soft bend. in EF-hand
true or false
false
its a sharp bend
calmodulin overview
CALMODULIN is a 17 kDa protein with a highly conserved amino acid sequence which contains 4 EF hands.
Following calcium binding it changes shape and is able to bind to and activate kinases.
is calcium ion an important secondary messenger invovled in muscle contraction
yes
does calcium ion control reease of hormones and neurotransmitters
yes
do many Ca+ bidning proteins contain an EF hand
yes
calmodulin isnt one of the main effectors of ca2+ signalling
true or false
false
it is one of the main effectors
is Ca+ and EF hand motif involved in binding of carbs by lectins
yes
what shape is calmodulin
dumbbell shape
where are the EF-hands on calmodulin
The N- and C-terminal domains each have 2 EF hands (EF1–4) separated by a unique 6-turn single a-helix.
do EF hands occur lone or in pairs
EF hands generally occur in pairs within globular domains and there is cooperative calcium binding.
apo definition
an apo structure is missing its ligand or binding partner
binding of calcium to calmodulin causes a conformational change
true or flase
true
exposing hydrophobic patch in each globular domain
what do ydrophobic patches in calmodulin and ca+ bidning allow
interactions with other proteins
hydrophobic patches binding target peptides
long helix of the dumb-bell unwinds and the two lobes of calmodulin swing around to enfold the a-helical target peptide.
A hydrophobic patch on each lobe of the calmodulin contacts the hydrophobic side of the helical target peptide.
Thus, the target peptide sits in a hydrophobic channel.
examples of target enzymes for calmodulin and ca+ binding
Phosphorylase kinase, myosin light chain kinase, adenylate cyclases and the Ca2+ ATP-ase.
Target enzymes have a mixture of basic and hydrophobic amino acids which are capable of adopting an a-helix.
how many EF hands does calmodulin have
4 each bind ca2+
is calmodulin a sensor
yes- is a sensor of an increase in intracellular [Ca2+], and calcium enhances its affinity for a number of important regulatory proteins.
