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In a single human cell there are between 10,000 and 100,000 coded messages known as genes. If all the directions contained in all these genes were written down, the words would fill the equivalent of 10,000 volumes of the Encyclopaedia Britannica.

Scientists at the Institute for Cancer Research in Philadelphia have bred mice that have more than one set of parents. Known as "multimice," these creatures are spawned by taking two embryos created by two sets of parent mice, placing them together in such a way that the embryos grow together, then transplanting the entire organism into the womb of a third female mouse. The result is a baby mouse born with genetic characterisitics of both set of parents.

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In a single human cell there are between 10,000 and 100,000 coded messages known as genes. If all the directions contained in all these genes were written down, the words would fill the equivalent of 10,000 volumes of the Encyclopaedia Britannica.

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Scientists at the Institute for Cancer Research in Philadelphia have bred mice that have more than one set of parents. Known as "multimice," these creatures are spawned by taking two embryos created by two sets of parent mice, placing them together in such a way that the embryos grow together, then transplanting the entire organism into the womb of a third female mouse. The result is a baby mouse born with genetic characterisitics of both set of parents.

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During experiments conducted in 1962 at the University of Michigan, scientists successfully extracted memory from one animal and transferred it to another. The experiment was conducted in the following manner. Over a period of time planarian worms were trained to behave in a particular way when exposed to light. These worms were then cut into pieces and fed to untrained planarians, and the untrained worms were put through the same learning paces as their predecessors. The second batch of worms, those that had dined on the first, learned many times faster than the originals, indicating that knowledge had somehow been transferred through body tissue. Similar experiments were later conducted at Baylor University: mice were trained to run through a maze, and an extract was then made of their brains. This extract was fed to untrained mice, which then learned the same maze twice as fast as their predecessors. If placed in a different maze, the untrained mice showed no particular aptitude for learning the layout. The implication of these experiments is that memory can be transferred from one being to another somatically as well as experientially.

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During experiments conducted in 1962 at the University of Michigan, scientists successfully extracted memory from one animal and transferred it to another. The experiment was conducted in the following manner. Over a period of time planarian worms were trained to behave in a particular way when exposed to light. These worms were then cut into pieces and fed to untrained planarians, and the untrained worms were put through the same learning paces as their predecessors. The second batch of worms, those that had dined on the first, learned many times faster than the originals, indicating that knowledge had somehow been transferred through body tissue. Similar experiments were later conducted at Baylor University: mice were trained to run through a maze, and an extract was then made of their brains. This extract was fed to untrained mice, which then learned the same maze twice as fast as their predecessors. If placed in a different maze, the untrained mice showed no particular aptitude for learning the layout. The implication of these experiments is that memory can be transferred from one being to another somatically as well as experientially.

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Testing the Chemiosmosis Theory

Several kinds of evidence support the chemiosmotic theory of ATP synthesis in chloroplasts.

Link to discussion of the chemiosmosis in chloroplasts.

(a) When isolated chloroplasts are illuminated, the medium in which they are suspended becomes alkaline — as we would predict if protons were being removed from the medium and pumped into the thylakoids (where they reduce the pH to about 4.0 or so).
(b) The interior of thylakoids can be deliberately made acid (low pH) by suspending isolated chloroplasts in an acid medium (pH 4.0) for a period of time. When these chloroplasts are then

• transferred to a slightly alkaline medium (pH 8.5), that is, one with a lower concentration of protons and
• given a supply of ADP and inorganic phosphate (P_i),

they spontaneously synthesize ATP. No light is needed.
Here is direct evidence that a gradient of protons can be harnessed to the synthesis of ATP.

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The Energy Relationships in Cellular Respiration and Photosynthesis: the Balance Sheet

The respiration (or burning) of a mole of glucose releases 686 kcal of energy. This value represents the difference between the energy needed to break the bonds of the reactants (glucose and oxygen) and the energy liberated when the bonds of the products (H₂O and CO₂) form.

Average bond energies, kcal/mole
C-H	98
O-H	110
C-C	80
C-O	78
H-H	103
C-N	65
O=O	116 (2 x 58)
C=O	187* (2 x 93.5)
C=C	145 (2 x 72.5)
(* as found in CO2)

Conversely, the photosynthesis of a mole of glucose requires the input of 686 kcal of energy.
The reasons:

• water and carbon dioxide
  • the differences in electronegativity between their atoms are high
  • so they have polar covalent bonds with high bond energies
  • these are strong bonds
    • broken with difficulty and
    • liberating copious amounts of energy when they form
• glucose and oxygen
  • the differences in electronegativity between their atoms tend to be lower
  • so they form covalent bonds with average bond energies on the low side
  • these are broken with relative ease

The diagram shows the details.
 

The overall equation for each process is the same; only the direction of the arrow differs. (The actual equation is:
but we shall ignore the six molecules of water that occur on each side as they "cancel out".)
The structural formulas are shown as well as the average bond energies for each bond involved.

Cellular Respiration

As you can see,

• the 24 moles of covalent bonds in a mole of glucose require a total of 2182 kcal to be broken.
• The 6 double bonds of oxygen require another 696.

As for the products,

• The formation of 6 moles of CO₂ involves the formation of 12 double polar covalent bonds each with a bond energy of 187 kcal/mole; total = 2244
• The formation of 6 moles of H₂O involves the formation of 12 O-H bonds each with an energy of 110 kcal/mole; total = 1320.

The minus sign indicates that free energy has been removed from the system.

Link to discussion of free energy.

Photosynthesis

The details of the energy budget are just the same. The only difference is that now it takes 3564 kcal to break the bonds of the reactants and only 2878 kcal are released in forming glucose and oxygen. So we express this change in free energy (+686 kcal) with a plus sign to indicate that energy has been added to the system. The energy came from the sun and now is stored in the form of bond energy that can power the needs of all life.

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Cellular Respiration

Index to this page

Mitochondria The Citric Acid Cycle The Electron Transport Chain Chemiosmosis in Mitochondria How many ATPs? Mitochondrial DNA (mtDNA)

Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water.

The energy released is trapped in the form of ATP for use by all the energy-consuming activities of the cell.
The process occurs in two phases:

• glycolysis, the breakdown of glucose to pyruvic acid
• the complete oxidation of pyruvic acid to carbon dioxide and water

In eukaryotes, glycolysis occurs in the cytosol. (Link to a discussion of glycolysis). The remaining processes take place in mitochondria.

Mitochondria

Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. Their number within the cell ranges from a few hundred to, in very active cells, thousands. Their main function is the conversion of the potential energy of food molecules into ATP.
Mitochondria have:

• an outer membrane that encloses the entire structure
• an inner membrane that encloses a fluid-filled matrix
• between the two is the intermembrane space
• the inner membrane is elaborately folded with shelflike cristae projecting into the matrix.
• a small number (some 5–10) circular molecules of DNA

This electron micrograph (courtesy of Keith R. Porter) shows a single mitochondrion from a bat pancreas cell. Note the double membrane and the way the inner membrane is folded into cristae. The dark, membrane-bounded objects above the mitochondrion are lysosomes.

The number of mitochondria in a cell can

• increase by their fission (e.g. following mitosis);
• decrease by their fusing together.

(Defects in either process can produce serious, even fatal, illness.)

The Outer Membrane

The outer membrane contains many complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out of the mitochondrion.

The Inner Membrane

The inner membrane contains 5 complexes of integral membrane proteins:

• NADH dehydrogenase (Complex I)
• succinate dehydrogenase (Complex II)
• cytochrome c reductase (Complex III; also known as the cytochrome b-c₁ complex)
• cytochrome c oxidase (Complex IV)
• ATP synthase (Complex V)

The Matrix

The matrix contains a complex mixture of soluble enzymes that catalyze the respiration of pyruvic acid and other small organic molecules.
Here pyruvic acid is

• oxidized by NAD⁺ producing NADH + H⁺
• decarboxylated producing a molecule of
  • carbon dioxide (CO₂) and
  • a 2-carbon fragment of acetate bound to coenzyme A forming acetyl-CoA

The Citric Acid Cycle

• This 2-carbon fragment is donated to a molecule of oxaloacetic acid.
• The resulting molecule of citric acid (which gives its name to the process) undergoes the series of enzymatic steps shown in the diagram.
• The final step regenerates a molecule of oxaloacetic acid and the cycle is ready to turn again.

Summary:

• Each of the 3 carbon atoms present in the pyruvate that entered the mitochondrion leaves as a molecule of carbon dioxide (CO₂).
• At 4 steps, a pair of electrons (2e^-) is removed and transferred to NAD⁺ reducing it to NADH + H⁺.
• At one step, a pair of electrons is removed from succinic acid and reduces the prosthetic group flavin adenine dinucleotide (FAD) to FADH₂.

The electrons of NADH and FADH₂ are transferred to the electron transport chain.

The Electron Transport Chain

The electron transport chain consists of 3 complexes of integral membrane proteins

• the NADH dehydrogenase complex (I)
• the cytochrome c reductase complex (III)
• the cytochrome c oxidase complex (IV)

and two freely-diffusible molecules

• ubiquinone
• cytochrome c

that shuttle electrons from one complex to the next.
The electron transport chain accomplishes:

• the stepwise transfer of electrons from NADH (and FADH₂) to oxygen molecules to form (with the aid of protons) water molecules (H₂O);(Cytochrome c can only transfer one electron at a time, so cytochrome c oxidase must wait until it has accumulated 4 of them before it can react with oxygen.)
• harnessing the energy released by this transfer to the pumping of protons (H⁺) from the matrix to the intermembrane space.
• Approximately 20 protons are pumped into the intermembrane space as the 4 electrons needed to reduce oxygen to water pass through the respiratory chain.
• The gradient of protons formed across the inner membrane by this process of active transport forms a miniature battery.
• The protons can flow back down this gradient only by reentering the matrix through ATP synthase, another complex (complex V) of 16 integral membrane proteins in the inner membrane. The process is called chemiosmosis.

Chemiosmosis in mitochondria

The energy released as electrons pass down the gradient from NADH to oxygen is harnessed by three enzyme complexes of the respiratory chain (I, III, and IV) to pump protons (H⁺) against their concentration gradientfrom the matrix of the mitochondrion into the intermembrane space (an example of active transport).

As their concentration increases there (which is the same as saying that the pH decreases), a strong diffusion gradient is set up. The only exit for these protons is through the ATP synthase complex. As in chloroplasts, the energy released as these protons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis and is an example of facilitated diffusion.
One-half of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their discovery of how ATP synthase works. Link to some of the details.

External Link

Animations of the electron transport chain and the workings of ATP synthase

Please let me know by e-mail if you find a broken link in my pages.)

How many ATPs?

It is tempting to try to view the synthesis of ATP as a simple matter of stoichiometry (the fixed ratios of reactants to products in a chemical reaction). But (with 3 exceptions) it is not.
Most of the ATP is generated by the proton gradient that develops across the inner mitochondrial membrane. The number of protons pumped out as electrons drop from NADH through the respiratory chain to oxygen is theoretically large enough to generate, as they return through ATP synthase, 3 ATPs per electron pair (but only 2 ATPs for each pair donated by FADH₂).
With 12 pairs of electrons removed from each glucose molecule,

• 10 by NAD⁺ (so 10x3=30); and
• 2 by FADH₂ (so 2x2=4),

this could generate 34 ATPs.
Add to this the 4 ATPs that are generated by the 3 exceptions and one arrives at 38.
But

• The energy stored in the proton gradient is also used for the active transport of several molecules and ions through the inner mitochondrial membrane into the matrix.
• NADH is also used as reducing agent for many cellular reactions.

So the actual yield of ATP as mitochondria respire varies with conditions. It probably seldom exceeds 30.

The three exceptions

A stoichiometric production of ATP does occur at:

• one step in the citric acid cycle yielding 2 ATPs for each glucose molecule. This step is the conversion of alpha-ketoglutaric acid to succinic acid.
• at two steps in glycolysis yielding 2 ATPs for each glucose molecule.

Mitochondrial DNA (mtDNA)

The human mitochondrion contains 5–10 identical, circular molecules of DNA. Each consists of 16,569 base pairs carrying the information for 37 genes which encode:

• 2 different molecules of ribosomal RNA (rRNA)
• 22 different molecules of transfer RNA (tRNA) (at least one for each amino acid)
• 13 polypeptides

The rRNA and tRNA molecules are used in the machinery that synthesizes the 13 polypeptides.
The 13 polypeptides participate in building several protein complexes embedded in the inner mitochondrial membrane.

• 7 subunits that make up the mitochondrial NADH dehydrogenase (complex I)
• cytochrome b, a subunit of cytochrome c reductase (complex III)
• 3 subunits of cytochrome c oxidase (complex IV)
• 2 subunits of ATP synthase (complex V)

Each of these protein complexes also requires subunits that are encoded by nuclear genes, synthesized in the cytosol, and imported from the cytosol into the mitochondrion. Nuclear genes also encode ~1,000 other proteins that must be imported into the mitochondrion. [More]

Mutations in mtDNA cause human diseases.

Mutations in 12 of the 13 polypeptide-encoding mitochondrial genes have been found to cause human disease.
Although many different organs may be affected, disorders of the muscles and brain are the most common. Perhaps this reflects the great demand for energy of both these organs. (Although representing only ~2% of our body weight, the brain consumes ~20% of the energy produced when we are at rest.)
Some of these disorders are inherited in the germline. In every case, the mutant gene is received from the mother because none of the mitochondria in sperm survives in the fertilized egg. Other disorders are somatic; that is, the mutation occurs in the somatic tissues of the individual.

Example: exercise intolerance

A number of humans who suffer from easily-fatigued muscles turn out to have a mutations in their cytochrome b gene. Curiously, only the mitochondria in their muscles have the mutation; the mtDNA of their other tissues is normal. Presumably, very early in their embryonic development, a mutation occurred in a cytochrome b gene in the mitochondrion of a cell destined to produce their muscles.
The severity of mitochondrial diseases varies greatly. The reason for this is probably the extensive mixing of mutant DNA and normal DNA in the mitochondria as they fuse with one another. A mixture of both is calledheteroplasmy. The higher the ratio of mutant to normal, the greater the severity of the disease. In fact by chance alone, cells can on occasion end up with all their mitochondria carrying all-mutant genomes — a condition called homoplasmy (a phenomenon resembling genetic drift).
Mutations in some 228 nuclear genes have also been implicated in human mitochondrial diseases.

Why do mitochondria have their own genome?

Many of the features of the mitochondrial genetic system resemble those found in bacteria. This has strengthened the theory that mitochondria are the evolutionary descendants of a bacterium that established anendosymbiotic relationship with the ancestors of eukaryotic cells early in the history of life on earth. However, many of the genes needed for mitochondrial function have since moved to the nuclear genome.
The recent sequencing of the complete genome of Rickettsia prowazekii has revealed a number of genes closely related to those found in mitochondria. Perhaps rickettsias are the closest living descendants of the endosymbionts that became the mitochondria of eukaryotes.

Further discussion of the evolutionary implications of mtDNA.

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Bioluminescence

Bioluminescence is the ability of living things to emit light. It is found in

• many marine animals, both invertebrate (e.g., some cnidarians, crustaceans, squid) and vertebrate (some fishes);
• some terrestrial animals (e.g., fireflies, some centipedes);
• some fungi and bacteria (photo at right)

The molecular details vary from organism to organism, but each involves

• a luciferin, a light-emitting substrate
• a luciferase, an enzyme that catalyzes the reaction
• ATP, the source of energy
• molecular oxygen, O₂

The more ATP available, the brighter the light. In fact, firefly luciferin and luciferase are commercially available for measuring the amount of ATP in biological materials.
Fireflies use their flashes to attract mates. The pattern differs from species to species. In one species, the females sometimes mimic the pattern used by females of another species. When the males of the second species respond to these "femmes fatales", they are eaten!

How Fireflies Control their Flashing

Barry Trimmer and his colleagues at Tufts University have recently discovered how fireflies turn their luminescent organs — called lanterns — on.

• The luminescent cells of the lanterns are close to cells at the end of the tracheoles (that bring oxygen to — and take carbon dioxide away from — the insect's tissues).
• These cells contain nitric oxide synthase (NOS), the enzyme that liberates the gas nitric oxide (NO) from arginine.

  Link to discussion of nitric oxide.

• Nerve impulses activate the release of NO from these cells.
• The NO diffuses into the lantern cells and inhibits cellular respiration in the mitochondria (probably by blocking the action of cytochrome c oxidase)
• With cellular respiration inhibited, the oxygen content of the cells increases.
• This turns on light production in the peroxisomes that contain luciferase and luciferin-ATP (the ATP is generated when the lanterns are dark).
• The quick decay of NO probably contributes to the short duration of the flash.

Bioluminescence in Marine Animals

The widespread occurrence of luminescence among deep-sea animals reflects the perpetual darkness in which they live.

• At least one fish has its luminescent organ located at the tip of a protruding stalk and uses it as bait to lure prey within reach of its jaws.
• When disturbed, one species of squid emits a cloud of luminescent water instead of the ink that its shallow-water relatives use.
• Some marine animals that live near the surface have luminescent organs on their underside. These probably make it more difficult for predators beneath them to see them against the light background of the surface.

In the case of fishes, the light is emitted by luminescent bacteria that grow in luminescent organs. The photos (courtesy of Prof. J. W. Hastings) show the flashlight fish, Photoblepharon palpebratus, with the lid of its luminescent organ open (left) and closed (right). The light is produced by continuously-emitting luminescent bacteria within the organs, but its display is controlled by the fish. These animals, which were photographed along reefs in the Gulf of Elat, Israel, appear to use their luminescent organs for such varied functions as

• attracting prey;
• signaling other members of their species; and
• confusing potential predators.

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ATP (Adenosine triphosphate)

ATP is a nucleotide that performs many essential roles in the cell.

• It is the major energy currency of the cell, providing the energy for most of the energy-consuming activities of the cell.
• It is one of the monomers used in the synthesis of RNA and, after conversion to deoxyATP (dATP), DNA.
• It regulates many biochemical pathways.

Energy

When the third phosphate group of ATP is removed by hydrolysis, a substantial amount of free energy is released. The exact amount depends on the conditions, but we shall use a value of 7.3 kcal per mole.
ATP + H₂O → ADP + P_i
ADP is adenosine diphosphate. P_i is inorganic phosphate. [structure]
Because of the substantial amount of energy liberated when it is broken, the bond between the second and third phosphates is commonly described as a "high-energy" bond and is depicted in the figure by a wavy red line. (The bond between the first and second phosphates is also "high-energy".) (But please note that the term is not being used in the same sense as the term "bond energy". In fact, these bonds are actually weak bonds with low bond energies.)

Discussion of bond energy.

Cells contain a wide variety of enzymes — called ATPases — that catalyze the hydrolysis of ATP and couple the energy released to particular energy-consuming reactions in the cell (see examples below).

Synthesis of ATP

• ADP + P_i → ATP + H₂O
• requires energy: 7.3 kcal/mole
• occurs in the cytosol by glycolysis
• occurs in mitochondria by cellular respiration
• occurs in chloroplasts by photosynthesis

Consumption of ATP

ATP powers most of the energy-consuming activities of cells, such as:

• Most anabolic reactions. Examples:
  • joining transfer RNAs to amino acids for assembly into proteins [Link]
  • synthesis of nucleoside triphosphates for assembly into DNA and RNA
  • synthesis of polysaccharides
  • synthesis of fats
• active transport of molecules and ions
• nerve impulses
• maintenance of cell volume by osmosis
• adding phosphate groups (phosphorylation) to many different proteins, e.g., to alter their activity in cell signaling.
• muscle contraction
• beating of cilia and flagella (including sperm)
• bioluminescence

Extracellular ATP

In mammals, ATP also functions outside of cells. Its release

• from damaged cells can elicit inflammation and pain;
• from the carotid body signals a shortage of oxygen in the blood;
• from taste receptor cells triggers action potentials in the sensory nerves leading back to the brain;
• from the stretched wall of the urinary bladder signals when the bladder needs emptying.

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The Urea Cycle

Urea is the chief nitrogenous waste of mammals.
Most of our nitrogenous waste comes from the breakdown of amino acids.
This occurs by deamination.
Deamination of amino acids results in the production of ammonia (NH₃).

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Ammonia is an extremely toxic base and its accumulation in the body would quickly be fatal.
However, the liver contains a system of carrier molecules and enzymes which quickly converts the ammonia (and carbon dioxide) into urea.
This is called the urea cycle.
One turn of the cycle:

• consumes 2 molecules of ammonia
• consumes 1 molecule of carbon dioxide
• creates 1 molecule of urea ((NH₂)₂CO
• regenerates a molecule of ornithine for another turn.

Although our bodies cannot tolerate high concentrations of urea, it is much less poisonous than ammonia.
Urea is removed efficiently by the kidneys.

Link to discussion of the anatomy and physiology of the kidneys.

What can go wrong?

There are several inherited diseases of the urea cycle caused by mutations in genes encoding one or another of the necessary enzymes.
The most common of these is an inherited deficiency of ornithine transcarbamylase, an enzyme needed for the conversion of ornithine to citrulline. It results in elevated levels of ammonia that may be so high as to be life-threatening.
It is an X-linked disorder; therefore most commonly seen in males. It can be cured by a liver transplant.
It can also be caused by a liver transplant! In 1998, an Austrian woman was given a new liver from a male cadaver who — unknown to the surgeons — had a mutation in his single ornithine transcarbamylase gene. The woman's blood level of ammonia shot up, and she died a few days later.

Uric acid

Humans also excrete a second nitrogenous waste, uric acid. It is the product of nucleic acid, not protein, metabolism. It is produced within peroxisomes.
Uric acid is only slightly soluble in water and easily precipitates out of solution forming needlelike crystals of sodium urate. These

• contribute to the formation of kidney stones;
• produce the excruciating pain of gout when deposited in the joints.

Curiously, our kidneys reclaim most of the uric acid filtered at the glomeruli. Why, if it can cause problems?

• Uric acid is a potent antioxidant and thus can protect cells from damage by reactive oxygen species (ROS). [Link]
• The concentration of uric acid is 100-times greater in the cytosol than in the extracellular fluid. So when lethally-damaged cells release their contents, crystals of uric acid form in the vicinity. These enhance the ability of nearby dendritic cells to "present" any antigens released at the same time to T cells leading to a stronger immune response.

So the risk of kidney stones and gout may be the price we pay for these protections.
Most mammals have an enzyme — uricase — for breaking uric acid down into a soluble product. However, during the evolution of great apes and humans, the gene encoding uricase became inactive. A predisposition to gout is our legacy.
Uric acid is the chief nitrogenous waste of

• insects
• lizards and snakes
• birds

(It is the whitish material that birds leave on statues.)
These animals convert the waste products of protein metabolism — as well as nucleic acid metabolism — into uric acid.
Because of its low solubility in water, these animals are able to eliminate waste nitrogen with little loss of water.

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Second Messengers

Second messengers are molecules that relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc. — to target molecules in the cytosol and/or nucleus.
But in addition to their job as relay molecules, second messengers serve to greatly amplify the strength of the signal. Binding of a ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.
There are 3 major classes of second messengers:

1. cyclic nucleotides (e.g., cAMP and cGMP)
2. inositol trisphosphate (IP₃) and diacylglycerol (DAG)
3. calcium ions (Ca²⁺)

Cyclic Nucleotides

Cyclic AMP (cAMP)

Some of the hormones that achieve their effects through cAMP as a second messenger:

• adrenaline
• glucagon
• luteinizing hormone (LH)

Cyclic AMP is synthesized from ATP by the action of the enzyme adenylyl cyclase.

• Binding of the hormone to its receptor activates
• a G protein which, in turn, activates
• adenylyl cyclase.
• The resulting rise in cAMP turns on the appropriate response in the cell by either (or both):
  • changing the molecular activities in the cytosol, often using Protein Kinase A (PKA) — a cAMP-dependent protein kinase that phosphorylates target proteins;
  • turning on a new pattern of gene transcription. [View mechanism]

Cyclic GMP (cGMP)

Cyclic GMP is synthesized from the nucleotide GTP using the enzyme guanylyl cyclase.
Cyclic GMP serves as the second messenger for

• atrial natriuretic peptide (ANP)
• nitric oxide (NO)
• the response of the rods of the retina to light. [Discussion]

Some of the effects of cGMP are mediated through Protein Kinase G (PKG) — a cGMP-dependent protein kinase that phosphorylates target proteins in the cell.

Inositol trisphosphate (IP₃) and diacylglycerol (DAG)

Peptide and protein hormones like

• vasopressin,
• thyroid-stimulating hormone (TSH), and
• angiotensin

and neurotransmitters like GABA
bind to G protein-coupled receptors (GPCRs) that activate the intracellular enzyme phospholipase C (PLC).
As its name suggests, it hydrolyzes phospholipids — specifically phosphatidylinositol-4,5-bisphosphate (PIP₂) which is found in the inner layer of the plasma membrane. Hydrolysis of PIP₂ yields two products:

• diacylglycerol (DAG)DAG remains in the inner layer of the plasma membrane. It recruits Protein Kinase C (PKC) — a calcium-dependent kinase that phosphorylates many other proteins that bring about the changes in the cell.
  As its name suggests, activation of PKC requires calcium ions. These are made available by the action of the other second messenger — IP₃.
• inositol-1,4,5-trisphosphate (IP₃)
  • This soluble molecule diffuses through the cytosol and
  • binds to receptors on the endoplasmic reticulum causing
  • the release of calcium ions (Ca²⁺) into the cytosol.
  • The rise in intracellular calcium triggers the response.Example: the calcium rise is needed for NF-AT (the "nuclear factor of activated T cells") to turn on the appropriate genes in the nucleus. [Discussion]
    The remarkable ability of tacrolimus and cyclosporine to prevent graft rejection is due to their blocking this pathway.

The binding of an antigen to its receptor on a B cell (the BCR) also generates the second messengers DAG and IP₃.

Calcium ions (Ca²⁺)

As the functions of IP₃ and DAG indicate, calcium ions are also important intracellular messengers. In fact, calcium ions are probably the most widely used intracellular messengers.
In response to many different signals, a rise in the concentration of Ca²⁺ in the cytosol triggers many types of events such as

• muscle contraction;
• exocytosis, e.g.,
  • release of neurotransmitters at synapses (and essential for the long-term synaptic changes that produce Long-Term Potentiation (LTP) and Long-Term Depression (LTD);
  • secretion of hormones like insulin
• activation of T cells and B cells when they bind antigen with their antigen receptors (TCRs and BCRs respectively)
• adhesion of cells to the extracellular matrix (ECM)
• apoptosis
• a variety of biochemical changes mediated by Protein Kinase C (PKC).

Normally, the level of calcium in the cell is very low (~100 nM). There are two main depots of Ca²⁺ for the cell:

• The extracellular fluid (ECF — made from blood), where the concentration is ~ 2 mM or 20,000 times higher than in the cytosol;
• the endoplasmic reticulum ("sarcoplasmic" reticulum in skeletal muscle).

However, its level in the cell can rise dramatically

• when channels in the plasma membrane open to allow it in from the extracellular fluid or
• from depots within the cell such as the endoplasmic reticulum and mitochondria.

Getting Ca²⁺ into (and out of) the cytosol

• Voltage-gated channels
  • open in response to a change in membrane potential, e.g. the depolarization of an action potential;
  • are found in excitable cells:
    • skeletal muscle
    • smooth muscle (These are the channels blocked by drugs, such as felodipine [Plendil®], used to treat high blood pressure. The influx of Ca²⁺ contracts the smooth muscle walls of the arterioles, raising blood pressure. The drugs block this.)
    • neurons. When the action potential reaches the presynaptic terminal, the influx of Ca²⁺ triggers the release of the neurotransmitter.
    • the taste cells that respond to salt [Link].
  • allow some 10⁶ ions to flow in each second following the steep concentration gradient.
• Receptor-operated channels
  These are found in the post-synaptic membrane and open when they bind the neurotransmitter. Example: NMDA receptors.
• G-protein-coupled receptors (GPCRs). These are not channels but they trigger a release of Ca²⁺ from the endoplasmic reticulum as described above. They are activated by various hormones and neurotransmitters (as well as bitter substances on taste cells in the tongue — Link).

Ca²⁺ ions are returned

• to the ECF by active transport using
  • an ATP-driven pump called a Ca²⁺ ATPase;
  • two Na⁺/Ca²⁺ exchangers. These antiport pumps harness the energy of
    • 3 Na⁺ ions flowing DOWN their concentration gradient to pump one Ca²⁺ against its gradient and
    • 4 Na⁺ ions flowing down to pump 1 Ca²⁺ and 1 K⁺ ion up their concentration gradients.
• to the endoplasmic (and sarcoplasmic) reticulum using another Ca²⁺ ATPase [Link].

How can such a simple ion like Ca²⁺ regulate so many different processes? Some factors at work:

• localization within the cell (e.g., released at one spot — the T-system is an example — or spread throughout the cell)
• by the amount released (amplitude modulation, "AM")
• by releasing it in pulses of different frequencies (frequency modulation, "FM")

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Serine Proteases

The serine proteases are a family of enzymes that cut certain peptide bonds in other proteins.

This activity depends on a set of amino acid residues in the active site of the enzyme — one of which is always a serine (thus accounting for their name).

In mammals, serine proteases perform many important functions, especially in digestion, blood clotting, and the complement system.

Digestive Enzymes

Three protein-digesting enzymes secreted by the pancreas are serine proteases [Link]:

• chymotrypsin
• trypsin
• elastase

These three share closely-similar structures (tertiary as well as primary). In fact, their active serine residue is at the same position (Ser-195) in all three.

Despite their similarities, they have different substrate specificities; that is, they cleave different peptide bonds during protein digestion. [More]

Clotting Factors

Several activated clotting factors are serine proteases, including

• Factors 10 (X), 11 (XI), and 12 (XII)
• Thrombin
• Plasmin

Complement Factors

Several proteins involved in the complement cascade are serine proteases, including

• C1r and C1s
• the C3 convertases
  • C4b,2a
  • C3b,Bb

Serpins

Serpins are Serine Protease Inhibitors.

Here is a list of a few important serine proteases and the serpins that control them.

Serine Protease	Serpin
Chymotrypsin	alpha-1-antichymotrypsin
Complement factor C1s	C1 Inhibitor (C1INH)
Elastase (secreted by neutrophils)	alpha-1-antitrypsin
Clotting factor 10 (X)	antithrombin III
Thrombin	antithrombin III
Plasmin	alpha-2-antiplasmin
Trypsin	pancreatic trypsin inhibitor

How Serpins Work

The serpins inhibit the action of their respective serine protease by mimicking the three-dimensional structure of the normal substrate of the protease.

• The serine protease binds the serpin instead of its normal substrate. This alone would block any further activity by the protease. But the serpin has another trick to play.
• The protease makes a cut in the serpin leading to
  • the formation of a covalent bond linking the two molecules;
  • a massive allosteric change in the tertiary structure of the serpin;
  • which moves the attached protease to a site where it can be destroyed.

Importance of Serpins

Almost 20% of the proteins found in blood plasma are serpins.

Their abundance reflects their importance: putting a stop to proteolytic activity when the need for it is over.

This is especially important for the

• clotting and
• complement

systems where a tiny initial activating event leads to a rapidly amplifying cascade of activity.

Serpin Deficiencies

A number of inherited human diseases are caused by a deficiency of a particular serpin. The deficiency usually results from a mutation in the gene encoding the serpin.

Examples:

Alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin inactivates the elastase secreted by neutrophils. When the lungs become inflamed, neutrophils secrete elastase as a defensive measure. However, it is important to inactivate this elastase as soon as its job is done. That is the function of alpha-1-antitrypsin.

(Its name, alpha-1-antitrypsin, suggests that it attacks the digestive enzyme, trypsin. In vitro, it does, but in the body, alpha-1-antitrypsin is found in the blood, not the intestine. Inactivation of trypsin in the intestine is the function of another serpin, pancreatic trypsin inhibitor.)

People with an inherited deficiency of alpha-1-antitrypsin (they are homozygous for a point mutation in its gene) are prone to emphysema. An effective treatment is on the horizon now that genetic engineering has produced goats that secrete human alpha-1-antitrypsin in their milk. [More]

Alpha-1-antitrypsin deficiency can also lead to liver damage. Alpha-1-antitrypsin is synthesized in the liver. However, some mutant versions of the molecule form insoluble aggregates within the liver cells. This mechanism is similar to that of the prion diseases where protein aggregates destroy neurons in the brain [Link]. A drug that enhances autophagy protects mice from the liver damage caused by aggregates of mutant alpha-1-antitrypsin.

C1INH deficiency

A deficiency of C1INH produces hereditary angioedema. Patients are at risk of occasional explosive triggering of the complement system. The massive release of anaphylatoxins (C3a, C5a) may cause dangerous swelling (edema) of the airways, as well as of the skin and intestine.

C1INH also inhibits kallikrein, the enzyme responsible for forming the potent vasodilator bradykinin. Hence, a deficiency of C1INH can trigger a violent inflammatory response by this mechanism as well. [More]

Antiplasmin deficiency

A deficiency in antiplasmin puts the person at risk of uncontrollable bleeding.

Antithrombin deficiency

A deficiency in antithrombin puts the person at risk of spontaneous blood clots, which can lead to a heart attack or stroke.

In January 2009, an advisory committee of the U.S. FDA decided that a recombinant human antithrombin (ATryn®) secreted into the milk of transgenic goats was safe for use in therapy.

The Evolution of the Serine Proteases

The close sequence similarity of the various mammalian serine proteases suggests that each is the product of a gene descended by repeated gene duplication from a single ancestral gene.

Other Serine Proteases

Serine proteases and molecules similar to them are found elsewhere in nature.

Subtilisin

Subtilisin is a serine protease secreted by the bacterium Bacillus subtilis. Although it has the same mechanism of action as the serine proteases of mammals, its primary structure and tertiary structure are entirely different.

Here, then, is an example at the molecular level of convergent evolution: two molecules acquiring the same function (analogous) but having evolved from different genes.

Acetylcholinesterase

This enzyme is built like and acts like the other serine proteases, but its substrate is the neurotransmitter acetylcholine, not a protein.

It is found at several types of synapses as well as at the neuromuscular junction — the specialized synapse that triggers the contraction of skeletal muscle.

The organophosphate compounds used as

• insecticides (e.g., parathion) and
• nerve gases (e.g. Sarin)

bind to the serine at the active site of acetylcholinesterase blocking its action.

Serpinlike Molecules

Angiotensinogen

This peptide is the precursor of angiotensin II — a major factor in maintaining blood pressure.

Link to discussion of angiotensinogen.

Chicken Ovalbumin

This is the major protein in the "white" of the egg (and a favorite antigen in immunological research).

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