protein mod and cell regulation 2

Question 1

what are the 4 phospholipid heads and there relative charges

Answer 1

phosphatidylserine negative
phosphatidylinositol negative
phosphatidylcholine neutral
phosphatidylethanolamine neutral

Question 2

what can happen to theses head groups if phosphorylated

Answer 2

become signalling molecules

Question 3

the internal monolayer of the plasma membrane is -ve because

Answer 3

of anionic phospholipids eg PS

Question 4

external monolayer is normally neutral because

Answer 4

it is composed of zwitterionic phospholipids

PC, PE and sphingomyelin

Question 5

distribution of the 2 leaflets is maintained by

Answer 5

flipases

Question 6

why is phospholipid asymmetry important

Answer 6

primary event in apoptosis is PS exposure - facilitates blood clotting
anionic surface

Question 7

why is phospholipid asymmetry important

bacteria

Answer 7

they have anionic membranes
different from ours
we produced selective cationic antibacterial proteins taht affects them and not us

Question 8

types of phospholipases

Answer 8

PLA1
PLA2- will relase arachidonic acid
PLC releases DAG and IP3
PLD releases phosphatidic acid

Question 9

key enzymes when discussing inflammation is

Answer 9

cytosolic PLA2

Question 10

cytosolic PLA2 is specific for

Answer 10

arachidonic acid at 
position 2 (sn-2)

Question 11

CPLA2 is a

Answer 11

cytosolic enzyme but translocates to membranes in the presence of (micro)M ca2+

Question 12

cPLA2 has 2 domains which are

Answer 12

N terminal C2 domain
ca2+ dependent phospholipid binding domain
c terminal domain containing the active site

Question 13

the cPLA2 enzyme is also a substrate for

activates the enzyme in vitro

Answer 13

MAP kinase

which phosphorylates at ser - 505

Question 14

the cPLA2 enzyme in cell culture is activated by

Answer 14

pro-inflammatory cytokines
eg interleukin 1
tumour necrosis factor and mitogens

Question 15

MAP kinase is also stimulated by

Answer 15

A23187 - ca ionophore

which increases cells [ca]

Question 16

ser-505 needs to be phosphorylated because

Answer 16

it goes into the membrane allowing the active site to open

Question 17

the entire activity of the Ca2+ binding is in

Answer 17

the C2 domain

Question 18

effect of binding Ca2+ to C2 domain is

Answer 18

it causes confomational change in the 3 Ca2+ binding loops

they become more hydrophobic

Question 19

2 of the loops that are now hydrphobic can

Answer 19

penetrate the bilayer providing a stable membrane protein interaction
prefer to bind to neutral membranes

Question 20

is phosphorylation important for activity

normal gene + s505A mutant

Answer 20

serine mutated to alanine

confirmed translocation with A23187 but failed to comoplete stimulation of arachidonic acid release

Question 21

what is believed that phosphorylation does

Answer 21

produces correct alignment of 2 domains on the membrane surface to produce maximal activity

Question 22

site of cPLA2 translocation
treated cells with A23187 translocate due to
and how do we know

Answer 22

increased [ca]
fixed cells and treat with rabbit ab to cPLA2
with fluorescent goat ab for rabbit igG

Question 23

where is it translocated

Answer 23

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

Question 24

c2 domains target specific membranes which are

Answer 24

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

Question 25

how is specific translocation achieved

Answer 25

A site specific docking or adaptor protein is required

enzyme colocalizes with COX-2