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SMOOTH ENDOPLASMIC RETICULUM
Textbook: pp. 324-327; (look at pp. 328-331 to further understand how centrifugation is
used cell biology); p. 232
The endoplasmic reticulum (ER) is organized into a netlike labyrinth of branching
tubules (mainly SER) amd flattened sacs (mainly RER) extending throughout the cytosol.
The tubules and sacs are interconnected, and their membrane is continuous with the outer
nuclear membrane. The space enclosed by this continuous membrane is called the ER
lumen. The sacs and tubes themselves are called cisternae (“reservoirs”) The ER lumen
often occupies more than 10% of the total cell volume and the ER membrane about 90%
of total cell membranes in many cells.. The functions of the SER include:
(1) Detoxification of xenobiotics e.g. pharmaceutical drugs. In mammals, this
occurs mainly in the hepatocytes (liver cells). The detoxification involves
the conversion of xenobiotics to more water soluble compounds, often by
the formation of hydroxyl groups. The enzymes that do this include
members of the so-called cytochrome P450 enzymes.
(2) Synthesis of steroid hormones. This synthesis requires the synthesis and
chemical modification of cholesterol. The cytochrome P450 enzymes are
are necessary for the modification of cholesterol.
(3) De novo synthesis of cholesterol
(4) Dephosphorylation of glucose-6-phosphate (glucose-6-P), especially in
liver cells, to allow efflux of glucose from the cells.
(5) Storage of Ca++ for intracellular signalling functions. This function is
especially evident in muscle cells, where the SER is called the
sarcoplasmic reticulum. (To be discussed when we talk about muscle
cells.)
(6) Synthesis of phospholipids for construction of membranes in the cell,
including those of the ER itself. Synthesis of bile acids
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Separtion of SER and RER by density gradient ultracentrifugation
The main way that the properties of the SER and RER have been elucidated is by
obtaining pure samples of SER and RER. This is done by;
(1) Breaking up the cells in an appropriate medium.
(2) Sedimentation of unwanted cell parts by centrifugation of
unbroken cells, nuclei, peroxisomes, lysosomes, and
mitochondria into a pellet, which is discarded.
(3) Placing samples of the supernatant on top of a sucrose density
gradient and centrifugation in an ultracentrifuge at about 150,000
x g. The SER and RER form small vesicles called microsomes
when they are shattered during the breaking up of the cells. The
SER microsomes are less dense than the RER microsomes
(because they do not have ribosomes attached to them).
With purified SER microsomes available, their biochemical and solute transport studies
can be studied.
Detoxification of xenobiotics
An important family of enzymes involved in
the detoxification of xenobiotics is the
cytochrome P450 family. The cytochromes
are proteins that have a heme group as a
prosthetic group. The heme group is attached
to its specific protein in various ways. Hemes
in different cytochromes can have different
side groups attached. Heme groups absorb
visible light and modifications of the heme
group by addition of different side groups, and
by attachment to different proteins, alters the
absorption spectrum. The P450 stands for
“pigment absorbing at 450 nm”.
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What you need to know about the heme group.
1) It is the prosthetic group of a range of very important proteins. (i) catalase (ii)
superoxide dismutase (iii) cytochrome complexes of the mitochondrial electron
transport system (iv) hemoglobin (vi) myoglobin (vii) the cytochrome P450 enzymes
of the SER.
2) Cytochromes reversibly carry O2 (hemoglobin and myoglobin) or perform redox
reactions that involve the reversible oxidation of the Fe ion bound to the heme.
Some heme groups use a Cu ion instead of a Fe ion.
3) Chorophyll is a heme group, using a Mg ion instead of Fe or Cu, with a long fatty
acid tail attached to make it soluble in the thylakoid membrane.
The cytochrome P450 complexes in the membrane of the SER consist of two enzymes:
(1) NADPH-cytochrome P450 reductase
(2) Cytochrome P450
The reductase enzyme does not contain a heme group.In each hydroxylation reaction,
two electrons are transferred from NADPH to the cytochrome P450 in a reaction
catalyzed by the NADPH-cytochrome P450 reductase. (Like NADH, NADPH is a
carrier for two electrons and one H+ in redox reactions.) while there is only one
reductase more than 100 different types of cytochrome P450 have been identified, each
with a different range substrate affinity. The overall hydroxylation reaction is:
Note that in this reaction the xenobiotic (R-H) has been oxidized by the addition of an
hydroxyl group that replaces a hydrogen atom. That is, the xenobiotic has been
hydroxylated. In the process the NADPH has also been oxidized, when it transfers its
two electron to the cytochrome P450 enzyme. Note that one atom of the diatomic the O2
molecule is reduced in the reaction to form water and the other O-atom becomes located
in the hydroxyl group that is added to the xenobiotic. The reaction is driven by the
electron flow resulting from the oxidation of the NADPH. The NADPH-cytochrome
P450 could have been called a NADPH dehydrogenase, but instead a name is given that
emphasizes that the enzyme reduces the cytochrome P450 (by reducing its heme group),
so it is called a reductase.
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NADPH + H+
NADP+
O2
ROH
H2 O
RH2
Fe3+
NADPH CYT P450
REDUCTASE
CYT P450
The hydroxylation of xenobiotics makes them more water-soluble and thus easier to be
carried in the bloodstream to the kidneys for excretion in the urine. Overall this system
“works” because it has been retained and elaborated during evolution. In other words its
possession must contribute to the likelihood of producing successful offspring.
However, for organisms that live well past their reproductive age there is some
downside to this system. Not only does the addition of hydroxyl, and related groups, to
make xenobiotics more water soluble. It can also convert them to more dangerous
compounds! An examples of this is the conversion of the benzopyrene in tobacco smoke
to a more potent carcinogen.
But there is also a “good side” to the “unthinking” addition of hydroxyl, and related
groups, to xenobiotics. That is the conversion of various pharmacuetical drugs to a
more active form. Two examples of this are:
(1) Conversion of various anti-depressants to a more active form.
(2) Conversion of codeine to morphine.
But life is never simple! It turns out that different human populations have different
amounts of each of the members of the cytochrome P450 family. So a drug that works
very well for one population make not work as well for another. Also, people within a
population also have different amounts of each of the cytochrome P450 enzymes. An
important case is that of the cytochrome P450 that recognizes codeine as a substrate. In
most people only about 5% of ingested codeine is converted to morphine, enough to
cause relief of pain but not enough to kill the person. Unfortunately, a small proportion of
people have up to 13 copies of the gene for this particular enzyme. This means they
convert codeine to morphine at a considerably faster rate in people with only two copies
of the gene.
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Even this faster rate of conversion does not seem to have a dangerous effect these
adults. But the situation becomes lethal when it is a pregnant mother that has the elevated
number of genes for the particular cytochrome P450 enzyme. There was a tragic
incidence in Toronto a few years ago when a mother with an elevated number of these
genes was given codeine. The baby died shortly after birth. There is now a whole new
field called pharmacogenetics which studies, amongst other things, the effect of variable
numbers of the various cytochrome P450 enzymes on the metabolism of drugs, both in
terms of beneficial and toxic effects.
Synthesis of steroid hormones
The synthesis of the steroid hormones is based upon the modification of cholesterol by
the SER. In mammals the mitochondria are also directly involved. Many of the
reactions involve the addition of hydroxyl groups (which may then oxidized to form
carbonyl groups). The cytochrome P450s are heavily involved in these processes.
Following are some aspects of steroid hormone synthesis, partly to emphasize the
combined involvement of two organelles: mitochondria and the SER. Cholesterol and
the, steroid hormones made from it, are highly insoluble in water. For this reason, the
enzymes involved in their biosynthesis, including the cytochrome P450 enzymes we have
already discussed, are located in membranes. They must be bound to sterol-binding
proteins to be able to move from the mitochondria to the SER, and from the SER to the
mitochondria.
1. In mammals the first step in steroid hormone synthesis involves the removal
of side chain located at the “top” of the cholesterol molecule. This is a complex
reaction performed by a cytochrome P450 enzyme located in the inner
membrane of the mitochondria. The reaction involves the addition of two
hydroxyl groups to the side chain to make its removal more chemically
favourable. See the figure entitled “Mitochondrial synthesis of pregnenolone
from cholesterol by mitochondria.” on the next page. (You need to be able to
draw the structure of cholesterol, but not the other structures. These other
structures are given to help you visualize what is going on.) All the steroid
hormones require the synthesis of pregnenolone.
2. Progesterone is synthesized in one step on the SER, after the pregnenolone
has left the mitochondrion. The synthesis of all the other steroid hormones
requires the synthesis of progesterone.
3. The synthesis of aldosterone shows how steroid hormone intermediates formed
on the SER may need to be transported back to the mitochondria for further
modification. (see the figure entitled “Involvement of the SER and the
mitochondria in the synthesis of aldosterone.”
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OH
OH
CHOLESTEROL
CYTOCHROME P450
PREGNENOLONE
CYTOCHROME P450
PROGESTERONE
MITOCHONDRIAL SYNTHESIS OF PREGNENOLONE FROM CHOLESTEROL
The first step catalyzed, by a cytochrome P450 in the inner membrane of the
mitochondrion is the addition of two hydroxyl groups to the cholesterol. Know the names
in the high-lighted boxes.
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PROGESTERONE
ALDSOSTERONE
INVOLVEMENT OF THE SER AND THE MITOCHONDRION IN THE SYNTHESIS OF ALDOSTERONE
REMEMBER that the goal in the last two diagrams is to show the need for both the
mitochondria and the SER in the biosynthesis of the steroid hormones, that
hydroxylations are involved (but not the chemical details), that hydroxylations are
performed by the cytochrome P450 enzymes, and that sterol-binding proteins are
needed to shuttle the water-insoluble intermediates between the mitochondria and
the SER, and vice versa.
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De novo synthesis of cholesterol
Sorry, but we do not have time to discuss the details of cholesterol biosynthesis in this
course! But I do want to emphasize, yet once again, how biosynthesis in eukaryotic
cells almost always involves enzymes located in more than one organelle. This means
also that transport systems are required to move biosynthetic intermediates between
the organelles.
3C
3C
PYRUVATE
GLUCOSE
PYRUVATE
NAD+
NADH + H+
ATP
ADP + Pi
CO2
CO2
PYRUVATE
DEHYDROGENASE
COA
2C
ACETYL-CoA
PYRUVATE
CARBOXYLASE
4C
OAA
CITRATE SYNTHASE
CITRATE
OAA
6C
4C
SER
MITOCHONDRION
CITRATE LYASE
CITRATE 6C
CoA
2C
ACETYL-CoA
CHOLESTEROL
SER ENZYMES OF CHOLESTEROL BIOSYNTHESIS
The complete oxidation of acetyl-CoAby the TCA cycle is clearly a catabolic reaction
( a “breaking down” reaction). But the TCA cycle also synthesizes compounds, for
example citrate, which are used for the biosynthesis of other compounds. In the
example at hand, citrate is required for the biosynthesis of cholesterol. The synthesis
of cholesterol is an anabolic reaction (a “building up” reaction). But if, as in this case,
citrate is being drained from the cycle, then their must be a replenishment of the OAA
to be able to join with acetyl-CoA to form more citrate. (OAA = oxaloacetate).
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The replenishment of OAA in the TCA cycle under these conditions is by the
carboxylation of pyruvate with CO2 in a reaction catalyzed by the enzyme pyruvate
carboxylase. This is an energy-requiring reaction which is driven by ATP hydrolysis.
Pyruvate carboxylase is located in the mitochondrial matrix. OAA formed in the
cytosol can also re-enter the mitochondrion in an indirect manner. Note that what was
really needed in the cytosol for cholesterol biosynthesis was not actually the citrate
the but the acetyl (acetate) group! I have used this example to show, once again, the
complex interactions that must occur between organelles! (Note: There are actually two
reactions required to convert the OAA to pyruvate in the cytosol, but for simplicity I have
not described them).
Biosynthesis of bile salts
Looking at the structures some of the bile salts one can easily guess that they are
biosynthesized from cholesterol i.e. cholesterol is their precursor. (Once again, you do not
have to learn these structures, but notice that they have a different number of O-atoms
than cholesterol and that the side chain has been shortened and had an amino acid
joined(“conjugated”) to it. These are the two most abundant bile acids.
Notice that, unlike the steroid hormones, the side chain is not completely cut off, it is
only shortened.. Instead the first step in a hydroxylation catalyzed by a cytochrome
P450.
A CYTOCHROME P450
We will not consider the further steps, but they are catalyzed by cytochrome
P450s and other enzymes in the SER membrane.
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And guess what, the peroxisomes are now known to be involved in the
biosynthesis of the bile acids! If you look at the structures the bile salts glycocholic and
taurocholic acid you see that an amino has been joined (“conjugated”) to a shortened
version of the sterol side chain. This addition is done in the peroxisomes. The amino
acid added to glycocholic acid is glycine. The actual addition of the amino acid does not
cause H2O2 production, but there is an involvement of an oxidase prior to this step that
occurs in the peroxisomes. Specific transport systems are required in the peroxisomal
membrane to move the intermediate from the SER and another transport system is
required to move the glycocholic and taurocholic acids out of the peroxisomes!
Dephosporylation of glucose-6-phospahte in liver cells
This topic is well described in your textbook (Fig. 17-23, reproduced below).
We store most of out glycogen in the liver cells (hepatocytes), but also
considerable amounts in our skeletal muscle cells. Here we discuss only the events in the
liver cells. We store glucose as glycogen because storing it has glucose itself would
have very severe osmotic and metabolic effects upon the hepatocytes.
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When we need to increase the flow of glucose into the bloodstream we increase the
rate at which the glycogen is broken down.
The relevant steps here are:
(1) Glycogen phosphorylase catalyzes the addition of inorganic phosphate
(Pi) to a terminal glucose units of glycogen chains to form soluble
glucose-1-P. This enzyme is known as a phosphorylase because the P that
is added is from inorganic phosphate ions. A kinase would have used the
terminal phosphate of ATP.
(2) The glucose-1-P is converted to glucose-6-P by phosphoglucomutase.
(3) The glucose-6-P is converted glucose and Pi by glucose-6-phosphatase.
This enzyme is located in the membrane of the SER. (The situation is
considerably more complex that is outlined in this diagram from your
textbook. The active site of the glucose-6-phosphatase actual faces the
lumen of the SER, not the cytosol as drawn in this diagram.(Think about
the consequences of the active site facing the SER lumen in terms of the
requirement for various transport systems.) People who have mutations in
this SER system for the conversion of glucose-6-P to glucose have a from
of diabetes.
(4) The glucose moves from the hepatocytes into the bloodstream via glucose
transport systems in the plasma membrane. These are facilitated
diffusion transport systems. Because of the rapid breakdown of glycogen
to glucose, the concentration of glucose in the hepatocytes becomes higher
than in the bloodstream, so glucose can move energetically downhill, as
long as there are hydrophilic gateways, the glucose transport systems, to
allow the hydrophilic glucose to cross the plasma membrane.