Sulfur Symbiosis

7/27/2019 Sulfur Symbiosis

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Sulfur Symbiosis:

Need O2, HS, CO2, Nitrogen Source (NO3) –used for AA production

HS SO3 APS SO4

This produces ATP and NADPH used to power Calvin Benson cycle.Mechanism:

HS and O2 bind reversibly to Hb.

Deliver these to symbion. HS then released from heme group and changed to SO3 by

sulfite reductase

APS (Adenosine phospho- sulfate) pathway, APS reductase and then APS sulfurase asin the smallest box, reducing Sulphate and can now be released.

Removing e (to ETC) , producing Energy (ATP) , & producing reducing equivalant

NADPH.

the symbion uses ATP & NADPH to power calvin cycle. fix carbon dioxide using rubisco(enzyme)

CO2 freely diffuses in, and the organism can setup a gradient for diffusion of CO2 byformation of bicarbonate (PH dependent

PH inside the riftia favors the conversion of CO2 to bicarbonate).

ultimately it gets fixed in the calvin benson cycle & produces food for itself & riftia.

2 method:1. translocation, release of organic molecules from the bacteria ,taken up by riftia2. digestion of bacteria that lives in them.

Alternative Mechanism of Carbon Fixation – Bristle Worm Episymbionts:

Reverse Citric Acid Cycle

ATP citrate-lyase Alpha-ketoglutaratsynthases (takes the place of alpha ketagluterate DH in

regular krebs)

Fumarate reductase

Cytochrome Oxidase of Riftia is Inhibited by HS



How does it deal with this?

1) Low rate of oxidative metabolis –important strategy for dealing with anoxia.

2) Perhaps HS is converted to a less toxic form for transport

3) Maybe there is a special blood horne HS- binding protein.

Mechanism is not known. Bacteria produce malate which is used by Riftia in oxidative pathway involving

cytochrome oxidase to generate ATP

Organisms require N for biosynthesis of AA and since bacteria are N fixing, they

are likely to also serve this function for Riftia.

Methane Exploitation

1) Methane monooxygenase2) Methanol dehydrogenase

Both of these produce the CH2O formaldehyde which goes into either the RuMP

cycle, Serine Cycle or RuBP.

Same process with HS, extraction of high energy e- oxidation of molecules,

adding to ETC, producing e motive force and using the e motive force to

synthesize ATP.

In the methanotropes e- enter from different points to the ETC.

Buffers

If Pka is close to the PH of the fluid it means there is 50% protonated acid and

50% of the base in the solution. Mean ing it could easily accept protons or –OH

on either side of the equation.

A buffer is an aqueous soln of a weak acid and its conjugate base (or a weak

base and conjugate acid), which resists the change in pH due to addition of a

strong base or strong acid. Can resist in both directions.

We need buffers for enzyme activity, the change in the state of protonation will

change the enzyme conformation and activity. Intracellular and extracellular buffers work together, as a change in the

intracellular buffer will be diffused into the extracellular fluid.

Anaerobic fermentation produces H+ but not by glycolysis! By ATP hydrolysis!

Acidification can limit survival during long-term anoxia.



Blood pH = 7.3-7.4, we would die outside of this range. Increase to 7.8 and you

get cramps and muscle tetanus (unable breath) and reduce it below 7 and you go

into coma.

pH outside of physiological range or optimum pH can:

- Inhibit enzymes or denature them- Lead to uncharged intermediates –which can freely defuse such as the Glucose-

Ps, neg charge intermediates can’t easily cross cell membrane.

- All organisms must have mechanisms to control pH

Two possibilities for dealing with H:

- Proton consuming rxn (AMP deaminase / ethanol production) – not immediate

reactions in the cell responding to pH change though!

- Proton or pH buffering (intracellular buffering) – immediate response to change,

simple buffers, rapidly remove protons from solution but don’t get rid of them.

Temporary. So the body have to remove it such as in bicarbonate system.- Proton consuming reactions are NOT WIDELY USED.

Two buffers:

1) Bicarb Buffer – requires CO2 to function well

a. Measure of blood CO2 or bicarb by partial pressure of CO2 gas.

b. Calculate or use method below in a closed chamber

c. Problem? This is an open system, CO2 can be released from the lungs or

urine. It is not a simple buffer system like phosphates are.

2) Non-Bicarb Buffers (phosphate, protein, dipeptide)

a. Measure total amount of each constituentb. Measure amount of acid or base required to change the pH of 1 g of tissue

by 1 pH unit –measurement of buffering power.

c. Unit is defined as the Van Slyke or beta.

Buffering power – how much acid you have to add to a solution to cause a

change in pH or how much does the pH change when you add acid or base to

soln.

The smaller the pH change, the greater the buffering power.

Focus on the Bicarb buffer system:

- 5% dissolved CO2

- 80% bicarb

- 15% carbamino compounds (Hb binding to CO2)

- Intracellular and extracellular buffer, CA is also found inside the RBC and

kidney cells along with the nephrons. The CO2 that is picked up is turned



into bicarb. Bicarb is released from RBC into the blood, for transfer to the

lungs.

- Mechanism is ONLY USEFUL IF OXYGEN IS PRESENT AND OXYGEN IS

RESPIRING! CO2 MUST EXIT AND O2 MUST COME IN!

In the tissue, metabolism producing CO2 and acid because of ATPases.CO2 can freely diffuse across membrane entering RBC.

CA will catalyze the hydration of CO2 into carbonic acid, H2CO3, which is a VERY

SHORT LIVED INTERMEDIATA that will dissociate into HCO3- and H+.

Bohr effect now deals with the acid, increase in partial pressure of CO2 or a decrease in

pH due to increase in H+ will reduce the affinity of Hb for O2.

So O2 is released, Hb binds the proton reducing the acidification.

Also Hb can bind CO2, becoming a carbamino hemoglobin on allosteric sites that

reduces the affinity of Hb for O2.

Halding Effect: deoxygenation of Hb increases the affinity of Hb for protons and CO2.

End product inhibition, limit of HCO3, transporter chloride bicarbonate antiporter

(BAN3), chloride comes in, bicarbonate goes to the blood.

In the lungs, everything is reversed. High O2 diffuses into the RBC and binds to Hbreducing the affinity of Hb to protons and CO2. Releasing CO2 and H+, CA catalyzes

the production of CO2 and H2O.

The proton becomes H2O and Co2 diffuses through the lungs to outside of the body.

Chloride bicarbonate antiporter works in reverse at the lungs, so the bicarbonate

will go into RBC, Cl out.

Greatest buffering potential is at pKa +/- 1 pH unit, thin this case its pKa is 6.1 so

the greatest buffering is from 5.1 to 7.1. Our blood is outside this range at 7.4.

Since we have an open system, CO2 can be removed or bicarbonate can be removed,

by the kidneys.



There is just so much more bicarb that can bind up the protons. If you increase CO2 at

tissue level, it is essentially acting as the acid because it pushes the rxn to the left

(production of H+). At the lungs, CO2 is being taken away rapidly so the rxn shifts to

CO2 production.

Not a simple buffer, it is OPEN at both ends. CO2 can increase its effectiveness, and

also high concentrations of bicarb in the blod (27 mM). 20x more than CO2 so more

protons being accepted.

Phosphate Buffer System (intracellular and extracellular):

- Plays a minimal role

- Pka is MUCH closer to physiological than bicarb system.

- H2PO4- H+ + HPO4 2-

- pKa 6.86 so effective from 5.86 to 7.86.

- Strong buffering power, effective from pH 6 to 8.

- Very small change in pH over a wide range of added base.

- But cellular [P] is only 1 to 2 mM this is insufficient, this is why it plays a MINOR

ROLE.

Protein Buffering (primarily intracellular)

If you have a single AA, depending on pH, the amine group can either accept or

release a proton as can the carboxyl group.

- Amino acids have a charged amine (+) and a carboxylate (-) group- Both can contribute to buffering

- Proteins are amino acid polymers composed of hundreds of these amino acids.

- However, a peptide bond masks any contribution to pH buffering by the groups,

therefore the side chains is the only group that can contribute to buffering!

Histidine is the ONLY AMINO ACID with a side chain in the physiological range (pKa is

6). This is still 1.4 pH below the physiological pH, so its not very good on its own as the

pKa of histidine changes depending on the micro-env around it.

Sometimes, the micro-env can give a pKa of 6.8 to histine.

Hb has 38 histidine residues, lots of buffering power!

Histidine has this buffering power because of its imidazole side group, can accept and

donate protons.



As we decrease the temperature, pH increases. Because H+ and OH- are being made

into H2O.

pH of blood follows that of pure water, in a closed system they parallel each otheras will

imidazole groups.

Neurtrality is when proton = hydroxyls, so the decreased temp tends tfor those ions to

form water = less of a tendency for water to break apart. pH is changed but neutrality

has not changed, but we measure pH with [H+]

Blood and cellular pH is defended in mammals even when temperature changes.

Cold-blooded animals allow pH to change with temperature, parallel to changes in pure

water, but maintains the ratio.

pH of blood is 7.4, we are essentially alkaline.

Increase the temperature and pH will decrease = more acidic. But [proton]/[hydroxyl]

ratio still the same.

An increase in temperature favours disscoation.

H-imid -> H+ + imid-

By definition at neutral pH or weak acids at the pK value the concentration of the

dissociation products must be equal.

Resulting in maintenance of alpha-stat at approx.. 50/50.

Alpha-Stat Hypothesis (Reeves)

If PH changes with temperature in parallel to changes in water, we will maintain a ratio

of imid groups.

pH of the cell and blood is actively regulated to maintain the fractional dissocation of

imidazole moiety at 50%. Perfect for a buffer, to maintain a 50% ratio, important for

enzymatic function.



Alpha imid = [imid-]/[imid-H+] + [imid-]

Allows for enzymes to function reversibily.

Alpha-stat hypothesis predicts pKa to be defended, not pH!

Organisms that use alpha stats, their pH changes (ectotherms) but they maintain

the alpha stat.

The phosphate buffer plays a small role, the proteins and peptides play the larger role.

His-195 gives catalytic site of LDH a pH sensitivity.

His is common to catalytic sites and therefore more enzymes are pH sensitive.

Fall in pH =increase in small H+, binding to His-195 of LD, changes conformation toincrease affinity for Pyr. Assuming presence of NADH.

Alpha stat allows the pH to changes while maintaining the ratio of protonated to

deprtonated groups due to temperature changes.

pH stat maintains pH regardless of temperature changes. Hypothesis will keep the pH

even if the temperature changes. The blood is acidotic, cause its neutral, water’s pH

has increase, but you have stopped the pH from increasing.

Cardiovascular Surgery – old vs new technique

To repair congenital heart defects in infants, surgeons combine the cardiopulmonary

bypass with deep hypothermia. Cooling the patient’s body temperature to 15 degrees.



Cold reduces the rate of metabolism prolonging the period in which the brain can be

safelty deprived of normal blood circulation.

The drop intemperature also makes the blood more alkaline (favours formation of H2O).

The normal pH level can be restored by adding carbon dioxide –to increase [H+]. Butthis has been controversial.

pH-stat is the adding of CO2 to blood to correct pH.

Comparative physiological studies carried out in the 1970-80s indicated that cold-

blooded vertebrates did not tend to compensate for temperature-related blood pH.

Surgeons questioned pH-stat because of this.

Studies in adults suggested that not restoring blood pH levels improved outcomes of

surgery. Switched to alpha-stat, leaving pH alone as neutrality is maintained.

pH protection:

- The pH stat strategy seems to protect the brain of a child.

- pH conditions under deep hypothermia appear to increase blood flow to thebrain.

- The addition of CO2 seems to further suppress the brain’s metabolism.

- Animal studies suggest that when the blood supply to the brain is limited, higher

CO2 levels are neuroprotective.

- Why doesn’t the pH stat improve outcomes in adults? Adult surgery is performed

under moderate hypothermia. So the change in blood pH is less dramatic.

- Additionally, the increase blood flow brought about by the pH strategy is morelikely to break off atherosclerotic plaque or clots, leading to a stroke.

Dipeptide Buffer System:

- Histidine containing dipeptides.



- Beta alanine –not a normal AA, allows the histidine to be immune to most of the

peptidases in body.

3 forms:

- Alanyl-his (carnosine)

- Alanyl-1-methylhis (anserine)- Anayl-3-methylhis (ophidine)

Major Correlation between amount of his-containing dipeptides and glycoltic rate of

muscle. Heart is very aerobic (slow-twitch, oxidizing non-glycolytic) = low

carnincine or anserine. Other muscles have high glycolytic rate = high

concentration of carnosine.

Chicken breast muscle = rarely used = glycolytic = lots of carnosine very important!

Correlation between anaerobic capacity and buffering capacity.

LDH activity= measure of glycolytic capacity of the muscle, high glycolytic capcity = high

buffering capacity.

Activity and glycolytic activity, clear correlation these are all white muscle, all glycolytic

muscle. Red muscle is aerobic.

In white muscle, the buffering capacity is twice as much as red muscle.

Legs used all the time = aerobic muscle

Dipeptide buffers do not seem important in non-mammals.

Marker enzymes = aerobic capacity (Citrate synthase) and Anaerobic capacity (LDH).

Fish have clearly defined red and white muscle. Mammals do not.

Invertebrate:



Inverts don’t seem to use His containing dipeptides. They have high concentration of

AAs tho.

Can’t just look at LDH as a measure of glylytic capcity. Have to look at all enzymes that

produce alternate end products.

They store massive amounts of PArg, which is used as a mechanism to buffer the

proton for PCr and PArg because PArg + ADP consumes a proton when transformed to

ATP and Arg.

The Shell as a buffer:

The shell is very mineralized, the use the shell in 2 ways:

- Can release carbonate (Ca or Mg carbonate) out to the blood

- Buffers Lact- and H+ forming HCO3- and CaLact+

- Lact- and H+ can also be stored in the shell and maintained there!

- Both occur but lactate sequestration in shell is very popular!

- This is what we do, we release minerals from bone

Other Buffers:

- Metabolic buffering, lactate to glucose or to CO2 and H2O.

- Organellar buffering

o . Lysosome pH changes with EC pH, so perhaps organelles like lysosome

can be involved in buffering some of those protons.

ROS and Antioxidants:

- Going from low level O2 to normoxia. How do deal with ROS.

- Wood frog nd turtle used as model organisms.

- ROS linked to reperfusion injury.

- ROS production is proportional to metabolic rate, mammals 1-4% of O2consumed is converted to ROS.

- There are 30+ O2 utilizing enzymes:

o Each one capable of generating ROS

o Cytochrome oxidase, xanthine oxidase, NADPH oxidase.



ROS have a BAD reputation, but recent research point out that they are required in

certain amount, involved in certain reversible reactions. 90% of O2 taken in is reduced

by cyt oxidiase at the end of ETC, so its clear if ROS production is related to metabolic

rate. Most ROs produced in mito.

UV light, metabolism, ionizing radiation, smoking can all lead to radicals that damage all

our macromolecules.

Three common ROS species:

- O2- superoxide

- H2O2 hydrogen peroxide

- H2O + OH* (hydroxyl radical)

Free e’s thought to be produced by electron leak from cytochromes ETC or other O2

utilizing reactions.

Hydroxyl Radical:

- Causes most damage in system to macromolecules.

- H2O2 requires a transition metal like iron.

- Producing hydroxyl ion and hydroxyl radical and ferric (3+).

- Ferrous is regenerated by the superoxide anion.- Cycle production of hydroxyl radical.

H2O2 + Fe2+ -> OH + OH* + Fe3+

O2- + Fe3+ -> O2 + Fe2+

Fenton reactions – transition metal catalyzed, other reductants can also make Fe2+

(GSH, ascorbate), Fe2+.

Fe2+ very reactive oxidant – body keeps the free ion low, it binds Fe2+ in hemes, to

reduce the effects of these reactive species.

Hydroxyl racical is Short-live (half-life is nanoseconds)



Other rxn also can regernate the iron, like vitamin C (ascorbate) which is an excellent

antioxidant. They won’t be involved in the Haber Weiss rxn but can regenerate the iron.

Exogenous Sources of Free radicals:

- Radiation – uv light, x-ray

- Chemical that react to form peroxides –ozone and singlet oxygen

- Chemical that promote superoxide formation Ub in ETC (quinones)

- Chemicals that are metbaolized to radicals (phenols, aminophenols), they can

also release iron and stimulate more hydroxyl radical by the haber-weiss.

- Chemicals that release iron –ferritin.

Endogenous Sources of ROS:

- All can produce ROS but main center is mito (90% of o2 intake reduced byCytOx).

- Cytoplasm – xanthine oxidase

- Peroxisome, oxidases

- Lysosomes, myeloperoxidase, phagocytes

- PM – lipoxygenases, NADPH oxidase – produces super oxide anions rapidly.

Kills the microorganisms, very useful.

Free Radical production by Ub in the mito inner membrane:

In the Q cycle, Ub donates e one at a time, so ubi-semi-quinol radical (intermediate) is

produced that can reduce O2 and produce superoxide anions (not very reactive but can

go using Haber-weiss to produce OH radical. This is an e- leak from ETC.

Originally when you use antimycin, increase in production of ROS because it blocks it at

the Q-cycle. However, if you use rotenone, it reduces the production of ROS because it

stops ETC at the NADH DH. At first, everyone though the most production came from

complex 3, but now we know comp 3 is the major produce of ROS as well as complexone (donates the 2- for Q which is part of complex 1).

Structure of Ub and Radical:



- This also works backwards as well. If you give up in e-, self-oxidize you release

a proton (remember the pumping H+ into IMS) an electrical and chemical

gradient.

Free Radical Damage:

- Oxidize SH (cysteine) –alter enzyme function - Cleavage of peptide bonds

- Modify carbohydrates

- Loss of metal from metalloproteins Fe2+ like heme from Hb

- DNA damage –breaks mutations

- Lipid peroxidation

Free radical theory of aging = over time the build of damage from the free radicals

leads to agigin, this is still partially believed, but many other factors involved.

Defense Systems against Free Radicals via Antiox:

1) Enzymatic defense:

a. Superoxide dismutase (SOD) –dismutation = oxidation and reduction of

the substrate in rxn

b. Glutathino peroxidase (GPOX)

c. Catalase (CAT)

2) Non-Enzymatic:

a. Glutathione (GSH)

b. Vitamine C (absorbic acid)

Enzymatic:

Super oxide anions:

O2- + O2- + 2H + --- SOD - H2O2 + O2 (one O2- oxidized, one reduced)

CAT can then take 2xH2O2 and change into 2H2O + O2.

2x GSH can be changed into GSSG using a H2O2 via GPOX.

GSSG can be reconverted to 2GSH at the cost of a NADPH via GSH reductase

- Regeneration of glutathione by gluthione reductase, NADPH donates a proton to

regenerate reduced gluthothione.



Hb-Fe2+ --H2O2- Hb-Fe3+

Fe2+ into Fe3+ this Fenton rxn is devastating because a methemoglobin, cannot bind

O2 lowering the O2 binding capacity in the blood.

Non-Enzymatic:

GSH (glutathione) most-abundant, tripeptide

Considered a “redox” buffer

Stress-tolerant organisms could then be said to have a high redox buffer capcity.

Why is this GSH not subject to degradation by protases? It has a gamma-peptide bond

which is resistant to enzymatic degradation by proteases. His is a good buffer but

organisms don’t store it in high concentration bc his is taken away to produce proteins,

but if they produce carnosine, it is not degraded and the imid group can function as a

buffer.

The carboxyl side chain of glutamate attached to an amine group of the cys = poly

gamma peptide bond.

Recall we talked about carnosine an uncommon AA (beta alanine) that produces a

strange peptide bond that most proteases can’t attack. Similar effect to GSH.

Glutathione made of Glutamine, cysteine and glycine SH between carboxyl side chain

of glu and amino of cysteine.

Reduced Glutathione can donate an e- reducing agent, can reduce ROS.

Oxidized Glutathione, joins with another GSH, production of disulfide bonds.



GSSG, GSH ratio is indicative of oxidative stress. GSH has had to donate lots of its e-

to O2.

Vitamin C absorbic acid, radical scavenger:

Ascorbic acid become dehydroacsorb acid.

Can donate H and become dehydro ascorbic acid.

Daily intake of Vit C only determined by its effect on collagen synthesis and not its

antiox. Perhaps we should take more!

Very effective antiox, can donate H-producing the double bonds.

Lipids are extremely susceptible to hydroxoyl radical damage.

3 steps:1) Initation of peroxidation

a. Hydroxyl radical produces a lipid radical, or fatty acid radical which reacts

with O2 to become a peroxyl radical

b. Poly unsat fatty acid in membranes most susceptible, double bonds leads

to more reactive ROS

2) Propagation

a. Lipid hydroperoxides, very damaging, changes the conformation of FA. 4

hydroxynanino, inhibits adenine nucleotide translocase as the effector of

the proton motive force and mess with ATP production.

b. Peroxyl radical that catalyze the chain rxn, can attack the poly unsaturatedFA next to it in membrane produing lipid hydropeoxide.

3) Termination

a. Antioxidants stop this and conver to non-radical products or stable

radicals.

Fluidity and ion channels are affected by this lipid peroxidation.



Consequences of Lipid Peroxidation:

- Structural changes in membrane:

o Alter fluidity and channels

o Alter membrane-bound signaling proteinso Increases ion peramebility

- Lipid peroxidation products form crosslinks with non-lipids

- Disruptions in membrane-dependent signaling

- DNA damage and mutagenesis

ATP -> Amp -> Adenonsine -> Inosine -> Hypoxanthine

Hypoxanthin in vivo, xanthine DH converts to Uric acid and NADH + H+ no ROS.

But when they grind the tissue, released Ca2+ activated protease which changes

xanthin DH into Xanthine oxidase which produces 2 superoxide radicals. This can be

stopped by a protease inhibitor.

Intracellular Ca2+ can increase due to ischemic/hypoxic/anoxic damage

triggering this conversion to xanthine oxidase. Reoxygenation injury.

Three Strategies:

1) Constitutively high antiox defense such as enzymes or GSH

2) Rapidly elevate antiox defenses in response to anoxic ischemic stress

3) Endure ROS damaged products post-insult –not really used bc its very

energy-consuming.

Good stress tolerant species choose 1. High antiox defenses!

High levels of GSH is observed in anoxia-tolerant species. Close to mammals, even

htough they are ecotherms. Close to mammals and bird because they have much lower

metabolic rates (ectotherms) but keep tese high to deal with stresses.

Extremely high level of reduced glutathione in anoxia tolerant organisms. GSSG:GSH

ratio increased = dealing with stress. GSH donates e- to form GSSG.



Levels of SOD, CAT and GPOX indicate tolerance to ROS.

They saw high enzymatic activity, constitutively in turtle. In anoxia intolerant organismdon’t maintain these high enzymatic activity.

In turtle, the enzymatic activity during anoxia changes very little, they anticipate it

coming. In garter snake it changes.

The idea of the body constantly fighting dangerous ROS has changed, as they are

produed in low levels to maintain homeostasis. Specially hydrogen peroxide

(H2O2 is an intracellular signalling molecule).

If they were only damaging, evolution would have dealt with it before.

Rapid changes in enzymatic activity, adaptation to stress has been shown to

increase in production of ROS. If you have lots of increase then they are

damaging.

General Survival Strategies

Good animal anaerobes or good stress tolerant animals.

Survival strategies or metabolic adaptations employed by organisms that tolerate stress:

METABOLIC RATE DEPRESSION:

1. Metabolic arrest

2. Channel arrest

Facultative anaerobes reduce the permability to these ions so the ATPases don’t have

to work as hard to maintain the gradient. Mammals can’t do this, leads to ischemic

death, apoptosis.



ATP turnover reduced significantly. Remember anoxic tolerant aimal show a reverse

Pasteur effect, reduce need for ATP when they sense low ATP levels. Don’t break as

much gly.

Metabolic Arrest:

- Purpose –maximizing survival time.

- Metabolic Arrest – specifically refers to the regulation of ATP producing

metabolic pathways, e.g. glycolysis and oxidative phosphorylation.

- Since anaerobic metabolism is primarily limited to the glycolytic generation of

lactate, glycolysis is the pathway of interest when referring to metabolic arrest.

- Has profound influence on all other survival strategies.

Metabolic arrest or a decreased metabolic rate will reduce the need for:

- More efficient fermentative pathways

- Increased substrate storage (glycogen)

- Increased buffering capacity (acidosis lactate)

- Minimizing Endproduct accumulation

- Utilizing proton consuming rxns

- Increasing antioxidant defenses.

Facultative anaerobiosis!

In general, rates for the arrested or dormant states are relative to the normoxic resting

metabolic rate at the same body temperature.

Characteristics of metabolic arrest:

A desirable regulatory mechanism for metabolic arrest would be:

1. Broadly applicable to all cell types and types of metabolic processes.

2. Readily coordinated as a response to a single extracellular signal.

3. Easily induce and readily reversed.

Generally, the metabolic transition to a depressed state probably shouldn’t involve gene

expression.But it happens, heat shock protein, stimulated during stress to deal with

issues after.



Mechanisms Investigated:

1. Enzyme-enzyme and enzyme-cytoskeletal assocations

2. Covalent Modification of key glycolytic enzymes

a. Phosphorylation or dephos leading to activation or inactivation of that

enzyme3. Allosteric Modification of key glycolytic enzymes

a. At a site other than the active site of the enzyme affecting the affinity of

the enzyme for its substrate.

Soluble enzymes are not free in the cytoplasm and don’t diffuse as they wish, enzymes

are organized to associate with one another especially if they are in the same pathway

–usually associated with CS elements.

1) Enzyme-enzyme or enzyme cytoskeletal associations

Enzymes that are localized along the myofibril hand-off product to their neighbour.

High flux = Pasteur effect enzymes are associated with myofibrils in low flux the

enzymes are no longer associated with actin.

Therefore, subsequent enzyme does not have to find its substrate.

Enzyme association and dissociation with each other or CS elements is mediated by

changes in cellular pH or covalent modification.

Experiment to determine partitioning of glycolytic pathway enzymes – homogenize and

centrifuge, if it is suspended in supernatant = not attached. If in pellet then its bound.

Anoxia intolerant species (mammal):

Enzyme activity – rest, work, ischemia

In rats leg, anoxia intolerant, at rest, the activity of the enzyme is found in supernata =

the majority of the enzyme are not bound to myofibrils.

In exercise, the majority of the enzyme is found in the pellet bound to myofibrils.

If you put the oragsnism under stress, ischemia, lack of O2, fuels and blood to the

tissue, majority of the enzyme are found bound and in the palette (Pasteur effect).



Anoxia tolerant animals show a reduced glycolytic rate and reduced binding of these

enzymes to myofibrils. pH stimulates the dissociation of the enzymes from the

myofibrils, so over the long term even anoxia tolerant organisms, the reduction in pH will

aid the reverse Pasteur effect making sure u don’t have high flux through glycolyticpathway.

Measure % bound of each of the glycolytic enzymes, in aerobic conditions, mostly

bound. After anoxia, much less enzymes are bound, in every single case significant

reduction of bound enzymes –reverse APsteur effect (rapidly inducible). After recovery

in most case you had the return to original aerobic level –reversible.

Decrease in pH will lead to lower flux of glycolysis because of enzyme

dissociations due to lower pH –lower pH seen in anoxia tolerant! Not in

mammals!

When

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