protein mod 2 opposite Flashcards
become signalling molecules
what can happen to theses head groups if phosphorylated
of anionic phospholipids eg PS
the internal monolayer of the plasma membrane is -ve because
it is composed of zwitterionic phospholipids
PC, PE and sphingomyelin
external monolayer is normally neutral because
primary event in apoptosis is PS exposure - facilitates blood clotting
anionic surface
why is phospholipid asymmetry important
they have anionic membranes
different from ours
we produced selective cationic antibacterial proteins taht affects them and not us
why is phospholipid asymmetry important
bacteria
PLA1
PLA2- will relase arachidonic acid
PLC releases DAG and IP3
PLD releases phosphatidic acid
types of phospholipases
cytosolic PLA2
key enzymes when discussing inflammation is
arachidonic acid at position 2 (sn-2)
cytosolic PLA2 is specific for
cytosolic enzyme but translocates to membranes in the presence of (micro)M ca2+
CPLA2 is a
N terminal C2 domain
ca2+ dependent phospholipid binding domain
c terminal domain containing the active site
cPLA2 has 2 domains which are
MAP kinase
which phosphorylates at ser - 505
the cPLA2 enzyme is also a substrate for
activates the enzyme in vitro
pro-inflammatory cytokines
eg interleukin 1
tumour necrosis factor and mitogens
the cPLA2 enzyme in cell culture is activated by
A23187 - ca ionophore
which increases cells [ca]
MAP kinase is also stimulated by
it goes into the membrane allowing the active site to open
ser-505 needs to be phosphorylated because
the C2 domain
the entire activity of the Ca2+ binding is in
it causes confomational change in the 3 Ca2+ binding loops
they become more hydrophobic
effect of binding Ca2+ to C2 domain is
penetrate the bilayer providing a stable membrane protein interaction
prefer to bind to neutral membranes
2 of the loops that are now hydrphobic can
serine mutated to alanine
confirmed translocation with A23187 but failed to comoplete stimulation of arachidonic acid release
is phosphorylation important for activity
normal gene + s505A mutant
produces correct alignment of 2 domains on the membrane surface to produce maximal activity
what is believed that phosphorylation does
increased [ca]
fixed cells and treat with rabbit ab to cPLA2
with fluorescent goat ab for rabbit igG
site of cPLA2 translocation
treated cells with A23187 translocate due to
and how do we know
to the perinuclear membrane
no translocation in control as they were not made Ca2+ permeable
COX is found in the same location within the cell
cox is the next enzyme in the formation of prostaglandin
where is it translocated
PC rich membranes - neutral - perinuclear membrane
plasma membrane is rich in PS
PKC also has a C2 domain but when ca binds it targets anionic PL found in the plasma membrane
c2 domains target specific membranes which are
A site specific docking or adaptor protein is required
enzyme colocalizes with COX-2
how is specific translocation achieved
