The “Direct Connection” section of MolBio Research Highlights includes blog posts discussing primary research articles in the field, but the interesting thing about it is that these posts are written by the authors themselves. This allows them to discuss the background, results and implications of their work with a wider audience and in a more relaxed format. Further, as it provides a direct link between the authors and the scientific community (hence its name), it promotes discussion. In today's "Direct Connection", Christopher Dieni, PhD, a postdoctoral scholar in the Department of Chemistry at Pennsylvania State University, discusses his recent publication entitled "Regulation of Glucose-6-Phosphate Dehydrogenase by Reversible Phosphorylation in Liver of a Freeze Tolerant Frog".
Sweet and Sour: Glucose-6-Phosphate, Anoxia, and Enzyme Regulation
A crazy model organism!
Grad school taught me a great many lessons, but I think many of them can be summarized in a brief and blunt statement: human beings aren’t that great!
Shocked?
Well, when you think about it, we’re not that cool of an organism at all. We’re extremely inefficient (biochemically speaking), and we’re really not that adaptable to… well… anything that perturbs our internal or external environment, however slight. By stark contrast, my graduate lab, or more accurately the lab of Dr. Ken Storey at Carleton University in Ottawa, Ontario, Canada, studies a wide array of awesome animals -model organisms- that can withstand harsh environmental conditions. These include frogs and snails that survive the desert heat and dehydration, mammals that hibernate over the long winter, reptiles and amphibians that can get by for months on an oxygen-deprived environment and my personal favorite and the central model organism of my graduate work, the freeze-tolerant wood frog, Rana sylvatica, that can survive the freezing of ~70% of its extracellular and extra-organ water during the winter, and then thaw in the spring and continue about its normal life.
A notable thing about the wood frog’s survival mechanisms is that one molecule plays a key role in keeping the frog alive during its frozen, unfathomable state: glucose. Most people find it hard to believe that this simple and ubiquitous six-carbon sugar can have such a prominent role in this extraordinary organism and yet, evolutionarily speaking, it makes perfect sense. Take a molecule with such universality and harness it maximally, not only for its biochemical and metabolic properties, but for its physical properties as well. High intracellular glucose can act as a cryoprotectant, as it prevents both intracellular freezing and the loss of intracellular water to the extracellular environment caused by the osmotic imbalance of extracellular freezing.
Wood frog, Rana sylvatica (Image from the Storey Lab)
Oh, so it’s that simple then!
It isn’t- nothing ever is. Clearly we’ve established that high concentrations of glucose are needed as a cryoprotectant, but we’ve also learned as far back as our first- or second-year biology or biochemistry courses how widely-used and widely-needed of a molecule glucose is. Glucose in fact, is also needed in frozen frogs -even if only a trickle- to keep a basal energy charge in order to maintain viability under these extreme conditions.
Among other things, when a frog freezes, it ceases breathing and becomes anoxic. When it thaws and suddenly begins breathing again, that rapid intake of oxygen can lead to the production of reactive oxygen species (ROS). Under these circumstances, ROS can potentially overwhelm the antioxidant defenses and so, their levels will need to be bolstered, as does the levels of nicotinamide adenine dinucleotide phosphatase (NADPH), which is needed for reducing power. The primary pathway, in turn, for generating NADPH is the pentose phosphate pathway, which draws upon -you guessed it- glucose-6-phosphate (G6P)!
So what’s the question here?
The question is clear: given this tug-of-war for glucose between cryoprotection, energy-consumption, and antioxidant defense, which one takes priority? There are several ways to approach this question experimentally (e.g. tracing metabolites, looking for changing levels of intermediates, etc.), but an interesting alternative way to discern how this metabolic flux is regulated, is to characterize the specific enzymes which catalyze the respective biochemical pathways in the particular physiological states. For example, we would analyze glycogen phosphorylase to see how glycogen is hydrolyzed to G6P in the liver, or glucose-6-phosphatase to gauge how G6P is dephosphorylated to glucose and subsequently exported to other organs. Further, studying phosphofructokinase would tell us how much G6P is being consumed in glycolysis, and if we wanted a clue about G6P being shunted through the pentose phosphate pathway, then characterizing the enzyme glucose-6-phosphate dehydrogenase (G6PDH) would be a good place to start.
Where to begin?
First, you need frogs in at least two groups: control and frozen. The control frogs were at 5 ºC and the frozen frogs were kept at -3 ºC. How long, you ask? Well, after 24 hours the frogs are known to be maximally frozen, so that’s typically the time point at which we sacrifice and dissect frozen frogs. That being said, it’s noteworthy that there are situations in which one wants to track metabolic changes as they occur in the freezing process. In situations like this, you’d probably want to sacrifice and dissect frogs over a time course, such as: 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, or something to that effect. Other metabolic changes can take place over the course of minutes rather than hours- it’s important to really have a feel for the model organism you’re working with and to cover all your bases.
Once we had the animals in the physiological states we wanted, we sacrificed and dissected them, and then flash-froze their individual organs in liquid nitrogen. Subsequently storing them in -80 ºC freezers allowed us to maintain the cellular contents in each of the physiological states until we’re ready to use them. Homogenizing tissues warrants careful attention, as one must be certain to preserve enzymes in their regulated state. Since phosphorylation, studied extensively for over 50 years, is the dominant post-translational modification of proteins, we hypothesized that any enzymes regulated in freeze-tolerance have a high probability of being in different phosphorylation states between the different physiological conditions. Thus, buffers used to homogenize the organs or tissues of interest must contain inhibitors of protein kinases and phosphatases in order to preserve the current phosphorylation state and prevent alteration. EDTA and EGTA chelate divalent cations like magnesium and calcium, which are essential to the activity of both kinases and phosphatases. Additional phosphatases inhibitors should be added and may include sodium fluoride, sodium pyrophosphate, sodium orthovanadate and β-glycerolphosphate, just to name a few. It should be noted that some of these inhibitors can also affect the assayable activity of your enzyme of interest. In fact, I’ve found sodium fluoride to be quite an effective (unwanted) inhibitor of some enzymes I’ve worked with.
After homogenizing and centrifuging, if at all possible, enzymes are assayed straight in the crude situation: with frogs averaging about 4 grams apiece, tissues are sometimes at a premium. G6PDH is a relatively easy enzyme to assay (at least in wood frog liver), as the substrates and products are simple to monitor and background isn’t a problem.
What to look for?
Once the assay conditions are optimized, a telltale sign are altered enzyme kinetic parameters. This is what we looked for in this publication and others. By gradually increasing the concentration of substrates and measuring the initial rate of reaction at each concentration, you can build an Activity v/s Substrate concentration profile and bang out kinetic parameters. Here, we saw that although the maximal catalyzed rate of reaction (Vmax) didn’t change between the control and frozen states of our frogs, the affinity of G6PDH for its substrates (Km) did. That alone, notably, was a clue that our enzyme of interest was being regulated.
Did that necessarily guarantee that the usual suspect, protein phosphorylation, had a hand in this? No. Differential kinetic parameters can be the result of post-translational modifications that include not only phosphorylation, but also many others, and can also derive from several other factors. Additional experiments were run to evaluate the role of phosphorylation further. We looked at the electrostatic properties of G6PDH, as revealed by its interaction with an ion-exchange column. The addition of each phosphate onto a protein adds up to 2 negative charges (I say “up to” because it’s always dependent on pH, surrounding protein environment, ionization constants, etc.), which will alter the net charge of the protein. It can also cause conformational changes which can bury and/or reveal other amino acid side chains, further altering the surface charge. Separating differently-charged forms of the enzyme suggests that G6PDH exists in a higher- and lower-phosphorylation state and these forms are interconverted during the shift between different physiological states of the frog.
Another nice experiment involves trying to “push” the phosphorylation state of the enzyme in one direction or the other using the frog’s own kinases and phosphatases and some biochemicals to stimulate them. Some signal transduction enzymes may co-purify with G6PDH, and we tried to stimulate activity from the more prominent ones.
For instance, adding cyclic AMP and ATP will help stimulate protein kinase A (PKA). Simply adding ions like magnesium and calcium will stimulate a plethora of phosphatases, though in our study, we couldn’t stimulate phosphatases possibly because they didn’t significantly co-purify with G6PDH or because they weren’t stable. Throughout all ion-exchange chromatography and stimulation of endogenous kinases and phosphatases, we looked for the same thing that we originally looked for in the crude situation: changing kinetic parameters of the enzyme and their corresponding patterns.
Fortunately, nowadays we have phospho-specific detection tools. By using western blots and a phosphoprotein stain, we learned that levels of total G6PDH weren’t changing between the different conditions, but its phosphorylation state was!
What have we learned?
We can say that based on our findings, differential phosphorylation of G6PDH may play a role in the frog’s transition to the frozen state. For one thing, it appears to cause a difference in the affinity by which the enzyme binds its substrates. What still presents a mystery, however (as we point out in the Discussion section of our paper), is that the change in Km may not be that significant in the face of increased G6P levels. What that means is that, despite lowered Km, given the elevated substrate concentrations, the enzyme can nonetheless easily find its way to maximal rates of reaction.
Future directions include the characterization of all the effects of reversible phosphorylation on G6PDH. We know that phosphorylation is changing in the face of freezing. We know that it’s doing something (spatial redistribution, maybe?). We just don’t fully grasp what it is yet, beyond what we’ve already observed. Each piece, of the puzzle, however, helps us understand this awesome animal and how it can do the incredible things that it does!
It isn’t- nothing ever is. Clearly we’ve established that high concentrations of glucose are needed as a cryoprotectant, but we’ve also learned as far back as our first- or second-year biology or biochemistry courses how widely-used and widely-needed of a molecule glucose is. Glucose in fact, is also needed in frozen frogs -even if only a trickle- to keep a basal energy charge in order to maintain viability under these extreme conditions.
Among other things, when a frog freezes, it ceases breathing and becomes anoxic. When it thaws and suddenly begins breathing again, that rapid intake of oxygen can lead to the production of reactive oxygen species (ROS). Under these circumstances, ROS can potentially overwhelm the antioxidant defenses and so, their levels will need to be bolstered, as does the levels of nicotinamide adenine dinucleotide phosphatase (NADPH), which is needed for reducing power. The primary pathway, in turn, for generating NADPH is the pentose phosphate pathway, which draws upon -you guessed it- glucose-6-phosphate (G6P)!
So what’s the question here?
The question is clear: given this tug-of-war for glucose between cryoprotection, energy-consumption, and antioxidant defense, which one takes priority? There are several ways to approach this question experimentally (e.g. tracing metabolites, looking for changing levels of intermediates, etc.), but an interesting alternative way to discern how this metabolic flux is regulated, is to characterize the specific enzymes which catalyze the respective biochemical pathways in the particular physiological states. For example, we would analyze glycogen phosphorylase to see how glycogen is hydrolyzed to G6P in the liver, or glucose-6-phosphatase to gauge how G6P is dephosphorylated to glucose and subsequently exported to other organs. Further, studying phosphofructokinase would tell us how much G6P is being consumed in glycolysis, and if we wanted a clue about G6P being shunted through the pentose phosphate pathway, then characterizing the enzyme glucose-6-phosphate dehydrogenase (G6PDH) would be a good place to start.
Where to begin?
First, you need frogs in at least two groups: control and frozen. The control frogs were at 5 ºC and the frozen frogs were kept at -3 ºC. How long, you ask? Well, after 24 hours the frogs are known to be maximally frozen, so that’s typically the time point at which we sacrifice and dissect frozen frogs. That being said, it’s noteworthy that there are situations in which one wants to track metabolic changes as they occur in the freezing process. In situations like this, you’d probably want to sacrifice and dissect frogs over a time course, such as: 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, or something to that effect. Other metabolic changes can take place over the course of minutes rather than hours- it’s important to really have a feel for the model organism you’re working with and to cover all your bases.
Once we had the animals in the physiological states we wanted, we sacrificed and dissected them, and then flash-froze their individual organs in liquid nitrogen. Subsequently storing them in -80 ºC freezers allowed us to maintain the cellular contents in each of the physiological states until we’re ready to use them. Homogenizing tissues warrants careful attention, as one must be certain to preserve enzymes in their regulated state. Since phosphorylation, studied extensively for over 50 years, is the dominant post-translational modification of proteins, we hypothesized that any enzymes regulated in freeze-tolerance have a high probability of being in different phosphorylation states between the different physiological conditions. Thus, buffers used to homogenize the organs or tissues of interest must contain inhibitors of protein kinases and phosphatases in order to preserve the current phosphorylation state and prevent alteration. EDTA and EGTA chelate divalent cations like magnesium and calcium, which are essential to the activity of both kinases and phosphatases. Additional phosphatases inhibitors should be added and may include sodium fluoride, sodium pyrophosphate, sodium orthovanadate and β-glycerolphosphate, just to name a few. It should be noted that some of these inhibitors can also affect the assayable activity of your enzyme of interest. In fact, I’ve found sodium fluoride to be quite an effective (unwanted) inhibitor of some enzymes I’ve worked with.
Frozen wood frog (Image from the Storey Lab)
After homogenizing and centrifuging, if at all possible, enzymes are assayed straight in the crude situation: with frogs averaging about 4 grams apiece, tissues are sometimes at a premium. G6PDH is a relatively easy enzyme to assay (at least in wood frog liver), as the substrates and products are simple to monitor and background isn’t a problem.
What to look for?
Once the assay conditions are optimized, a telltale sign are altered enzyme kinetic parameters. This is what we looked for in this publication and others. By gradually increasing the concentration of substrates and measuring the initial rate of reaction at each concentration, you can build an Activity v/s Substrate concentration profile and bang out kinetic parameters. Here, we saw that although the maximal catalyzed rate of reaction (Vmax) didn’t change between the control and frozen states of our frogs, the affinity of G6PDH for its substrates (Km) did. That alone, notably, was a clue that our enzyme of interest was being regulated.
Did that necessarily guarantee that the usual suspect, protein phosphorylation, had a hand in this? No. Differential kinetic parameters can be the result of post-translational modifications that include not only phosphorylation, but also many others, and can also derive from several other factors. Additional experiments were run to evaluate the role of phosphorylation further. We looked at the electrostatic properties of G6PDH, as revealed by its interaction with an ion-exchange column. The addition of each phosphate onto a protein adds up to 2 negative charges (I say “up to” because it’s always dependent on pH, surrounding protein environment, ionization constants, etc.), which will alter the net charge of the protein. It can also cause conformational changes which can bury and/or reveal other amino acid side chains, further altering the surface charge. Separating differently-charged forms of the enzyme suggests that G6PDH exists in a higher- and lower-phosphorylation state and these forms are interconverted during the shift between different physiological states of the frog.
Another nice experiment involves trying to “push” the phosphorylation state of the enzyme in one direction or the other using the frog’s own kinases and phosphatases and some biochemicals to stimulate them. Some signal transduction enzymes may co-purify with G6PDH, and we tried to stimulate activity from the more prominent ones.
For instance, adding cyclic AMP and ATP will help stimulate protein kinase A (PKA). Simply adding ions like magnesium and calcium will stimulate a plethora of phosphatases, though in our study, we couldn’t stimulate phosphatases possibly because they didn’t significantly co-purify with G6PDH or because they weren’t stable. Throughout all ion-exchange chromatography and stimulation of endogenous kinases and phosphatases, we looked for the same thing that we originally looked for in the crude situation: changing kinetic parameters of the enzyme and their corresponding patterns.
Fortunately, nowadays we have phospho-specific detection tools. By using western blots and a phosphoprotein stain, we learned that levels of total G6PDH weren’t changing between the different conditions, but its phosphorylation state was!
What have we learned?
We can say that based on our findings, differential phosphorylation of G6PDH may play a role in the frog’s transition to the frozen state. For one thing, it appears to cause a difference in the affinity by which the enzyme binds its substrates. What still presents a mystery, however (as we point out in the Discussion section of our paper), is that the change in Km may not be that significant in the face of increased G6P levels. What that means is that, despite lowered Km, given the elevated substrate concentrations, the enzyme can nonetheless easily find its way to maximal rates of reaction.
Future directions include the characterization of all the effects of reversible phosphorylation on G6PDH. We know that phosphorylation is changing in the face of freezing. We know that it’s doing something (spatial redistribution, maybe?). We just don’t fully grasp what it is yet, beyond what we’ve already observed. Each piece, of the puzzle, however, helps us understand this awesome animal and how it can do the incredible things that it does!
___
Reference
Dieni CA, & Storey KB (2010). Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog. Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology PMID: 20532891
(The "Direct Connection" logo is derived from Int. J. Mol. Sci. 2009, 10, 2763-2788)
3 Comments:
Many thanks to Alejandro for getting this up and running. It was a pleasure to write!
-- Chris
Really interesting, and it's a great idea to get article authors to write about an article :) It was interesting to hear of the glucose system being used to cope with extreme cold, will have to check if the ice-bound bacteria use a similar system.
Hi Lab Rat, thanks for the great comments! If you're looking for freeze-tolerant systems in bacteria, then you might be interested in this Naturwissenschaften 2007 review article: Cold-loving microbes, plants, and animals- fundamental and applied aspects. Ken Storey, my PI from grad school, is the last author. Check it out... it might be helpful. http://www.springerlink.com/content/20246821035501j0/
-- Chris
Post a Comment