© Whole Foods Magazine
An Interview with ATP Pioneer Dr. Eliezer Rapaport
Richard A. Passwater
In several of my recent columns, I have been discussing the importance of improving the body’s ATP level to optimize health. I have discussed how important ATP is for life and health, and dietary ways to improve our ATP levels. One particularly important column was with Dr. Steve Sinatra in which he concluded, “It’s all about ATP.” Dr. Sinatra was referring particularly to cardiovascular disease, but now I want to follow up this discussion with why health in general is so dependent on ATP.
This is the first of several interviews with World-renowned ATP expert Dr. Eli Rapaport. In this first chat, we will discuss what ATP is, what it does and why it is important to your health.
From my past writings, you may have detected that ATP is one of my favorite topics. I have been studying ATP almost as long as I have been researching antioxidants and selenium. However, my interest in ATP goes back to the days of my youth. Please let me explain with a little story.
Did you catch fireflies when you were very young? I did, only we used to call them “lightening bugs.” Before the days of air conditioning, we used to play outdoors most of the time during school vacation – baseball during the sunlight and street games like hide and seek etc. in the evening. It used to be too hot in upstairs bedrooms to sleep comfortably, so we would often sleep on the porch. Before settling down for the night, we would catch lightening bugs and put them in a Mason preservation (canning) jar in which we poked holes in its lid with an ice pick. Yes, an ice pick because we had no refrigerator and were dependent on the ice man delivering 25 pounds of ice for our ice box everyday to cool foods. The milkman would not only deliver milk, but go around back, open the kitchen door before we were up, and put the milk in the ice box. (No, the milk was not homogenized in those days, so the cream would separate and rise to the top.)
I bet not many of you used Mason jars for their intended purpose of making preserves from the foods we grew in our “Victory gardens.” Nearly everyone with a patch of dirt raised vegetables in their victory gardens during World War II.
As night would fall and while my parents gathered around the radio (not every home had a radio then), I would be watching Nature. I would wonder about things such as what caused the cooling breezes or what purpose those pesky mosquitoes served that nicer bugs couldn’t provide. Another persistent question was what purpose did the firefly lantern serve? What magic made it light? Why did the lanterns blink instead of just staying on?
Several years later, about 1950, Dr. Bill McElroy at Johns Hopkins was paying a penny a firefly if you brought him a jar with a couple hundred or so. As a teenager, I was too old for catching lightening bugs, but if that deal had only been available when I was younger, I could have been rich as a child. However, I would have had to talk my older brother into driving down US Route 40 to Baltimore! Gas was only 15 cents a gallon then, but I doubt that our 16-year old family car could have survived the 70-mile trip.
Dr. “Mac” McElroy became one of the most respected biochemists of all time. Now, why would this prestigious scientist pay a penny for a lightening bug? What was so important about the magic of the firefly glow?
Life needs energy in order to exist. Most biochemical reactions require an external source of energy in order to proceed. The energy that drives the reactions of life must be released gradually and on demand. It must be stored in readily accessible forms and the rate of energy release must be finely controlled.
In 1929 Fiske and Subbarow in Boston, discovered, characterized and identified ATP as the molecule involved in muscle contraction, though others, mostly Lohmann in Europe were unjustifiably credited with this discovery. In 1941, Lipmann identified ATP as the “high-energy” molecule in metabolism, and in 1947, Dr. McElroy determined that ATP was involved in the Firefly glow. However, ATP alone could not be the entire energy source for light emission since the energy released by ATP hydrolysis (7 kcal) is far less than the energy of a photon in the yellow region of the spectrum (50 kcal).
In addition to my antioxidant research, I was developing and publishing research about fluorometric and other luminescence applications. I had moved to Silver Spring, MD to head a research applications laboratory for the American Instrument Company and to continue my research with antioxidants and luminescence. Bioluminescence was now a possible tool for studying many biochemical pathways. We developed an instrument called the Aminco Chem-Glo which used measured ATP. Even today, I keep tabs with a close friend, Dr. David Miller, who continues to develop bioluminescence techniques and instrumentation.
When I met Dr. Bill “Mac” McElroy in 1965, the research group at Johns Hopkins was still trying to solve the problem. Later, I would be introduced to other members of his group including Drs. Howard Seliger, Nathan Kaplan and E. H. White who had joined Dr. McElroy’s staff in the late 1950s. In 1965, Drs. McElroy and Seliger published a seminary paper linking the evolutionary origins of bioluminescence with the appearance of oxygen in geological time. Dr. McElroy was promoted Chair the Biology Department at Hopkins and later became the Chancellor at the University of California, San Diego. Dr. Kaplan moved with him.
Now why am I telling you this long story about Johns Hopkins research with ATP and Dr. McElroy?
Drs. Seliger and White continued the quest to solve the Firefly mystery. Now entering Dr. White’s laboratory at the department of chemistry is a bright young grad student by the name of Eliezer Rapaport. That’s right, the same Dr. Rapaport we will chat with now.
Passwater: Is my recollection about Johns Hopkins and Fireflies fairly accurate?
Rapaport: Yes. It is correct and vivid. I studied at The Johns Hopkins University Graduate School for a Ph.D. in Chemistry and during these years we established the mechanism of the firefly bioluminescence (J. Amer. Chem. Soc. 91:2178-2180, 1969).
Passwater: And, that was the chemical basis for the Aminco Chem-Glo which I mentioned in the introduction. It lead to elucidating many, many enzymatic biochemical systems using your basic discovery. What got you interested in biochemistry in the first place?
Rapaport: I was initially interested in synthetic and mechanistic organic chemistry. The firefly bioluminescence was a perfect problem for these interests. Only later, after completing my studies at Hopkins, I moved to Harvard Medical School where I became interested in medical and physiological problems.
Passwater: Why did you want to study Firefly bioluminescence at Hopkins?
Rapaport: Hopkins was the place where bio- and chemiluminescence reactions were being studied. These type of chemical or biological (enzyme-catalyzed) reactions yield an electronically excited state product. The product emits light or photons in its return to an electronically ground state. Overall chemical energy is converted to light.
Passwater: What did you see that led the team to solving the long sought after question?
Rapaport: We had to identify the product of the firefly light reaction, the product being chemically unstable. The starting materials for the firefly bioluminescence were well established, firefly luciferin, ATP and the enzyme luciferase. Once we identified the reaction product, we established the reaction mechanism leading to the enzyme-bound product in an electronically excited state followed by the yellow-green light emission.
Passwater: My first books were a 3-volume "The Fluorescence Guide to the Literature" by Plenum Press and I was the editor of Fluorescence News. It seemed to me that the most practical and sensitive tests in the world for countless biochemical analysis could use your luciferin/luciferase technique, so I became very interested in your research.
Rapaport : Little did I know then that the light reaction of firefly luciferin, catalyzed by firefly luciferase and dependent on ATP would turn out to be the most sensitive and commonly used method for determining ATP levels as you mentioned. In the presence of excess luciferin, the enzyme luciferase and in the presence of limiting concentrations of ATP, the rate of the overall firefly light reaction is dependent on its first step, the formation of luciferyl adenylate. Measurement of the light intensity directly correlates with the amount of ATP.
Passwater: You extended this information into various directions. When did you move on to Harvard and what did you pursue there?
Rapaport: In 1971, I moved to Harvard Medical School where I started looking at the mechanisms of intracellular ATP functions, especially in relation to tumor growth and in vivo cancer models. The thought then was that because of its metabolic lability (ability to change), ATP is not only the energy currency molecule, a substrate for biosynthetic reactions and an allosteric regulator of protein functions, but an intracellular signal, the levels of which could change quickly in response to extracellular events.
Extracellular signals were thought to be stable molecules such as peptides or steroid hormones, since they had to survive the catabolic activities inside the vascular bed. Today we know that ATP and its catabolic product, adenosine, are overwhelmingly the two most "popular" extracellular signals, exactly because of their metabolic lability. Their metabolic lability enables them to act quickly only at sites and times as needed. ATP and adenosine interact extracellularly with families of receptors (P2X and P2Y for ATP and A for adenosine) and transmit signals, which are transduced into the inside of the cell. Through these mechanisms ATP and adenosine control virtually every physiological function.
Passwater: That’s very important and we’ll certainly pick up on that point later. For right now, though, please give us an example.
Rapaport: When one gets a cut in the skin and starts bleeding, the first event is the formation of a white plug or platelet aggregation. Such an event has to be induced by a labile molecule since one would not want a clot to be formed along the whole vessel, just at the site of the bleeding. The initial signal for recruiting platelets to aggregate, which is the first step in clot formation, is ADP (adenosine 5’-diphosphate), a molecule as labile as ATP and interacting with an ATP receptor on platelets. I will take a momentary leap here and describe how such receptors can be exploited in drug development. Since intravascular clotting, being a major cause of stroke and myocardial infarction, is highly undesirable, one way to block it is by antagonizing ADP binding to its platelet ATP receptor. A popular anti-thrombotic drug called Plavix or Clopidogrel achieves that by binding to the P2Y12 receptor on platelets, blocking the binding of ADP and reducing the possibility of intravascular platelet aggregation. Extracellular ATP today is known to signal diverse functions such as the control of breathing, interacting with peripheral and central chemoreceptors as well as to signal the transmission of taste from the taste buds to the central nervous system, thus being the neurotransmitter linking taste receptors to taste nerves. Evolution captured the most popular intracellular molecule to fulfill the most diverse extracellular signaling, once multicellular organisms were formed.
Passwater: So, ATP is more than the body’s energy currency. Its signaling function is also important. However, before we discuss more of the importance of ATP, let’s go back to the beginning and look at ATP basics.
Rapaport: You are right, "It is all about ATP". Ironically, intracellular ATP is making a powerful comeback, as its inverse relationships to aging are being unraveled. The significant decline in skeletal muscle ATP synthesis is acknowledged today as being causal in aging. OK, let’s look at ATP 101 for our readers.
Passwater: Let’s start with “What is ATP?”
Rapaport: ATP is an organic molecule comprised of a purine (adenine) moiety attached through a 9-1’ glycosidic linkage to a D-ribose sugar and three phosphate units esterified to the 5’ hydroxyl of the ribose. (Please see Figure 1.) The high-energy bond of ATP is the terminal pyrophosphate bond of the beta to gamma phosphate groups. The chemical energy of the energy-rich phosphate bond of ATP can be transferred to other cellular materials or be converted to mechanical energy, as is the case during the process of muscle contraction.
Figure 1. Chemical structure of Adenosine triphosphate (ATP)
Passwater: What does ATP do?
Rapaport: Intracellularly, ATP is:
· the major cellular and organ energy source
· a phosphate and adenylyl groups donor
· an intermediate in numerous cellular biosynthetic reactions
· a regulator of the activity of a variety of cellular proteins.
Extracellularly, ATP and its in vivo catabolic product, adenosine, regulate intracellular reactions in every organ and tissue by interacting with their specific families of receptors.
Passwater: How does ATP do this?
Rapaport: ATP as the energy currency acts by releasing the energy locked in the hydrolysis of the beta-gamma phosphodiester bond, yielding adenosine 5’-diphosphate (ADP). As a phosphate donor ATP transfers the gamma phosphate group to hydroxyl groups of the amino acids serine or tyrosine on proteins in a class of reactions catalyzed by enzymes called kinases. The phosphorylation of proteins is a major regulatory step inside the cell. ATP acts as a phosphate donor to a great variety of low molecular weight molecules containing hydroxyl, sulfhydryl and amino groups in reactions catalyzed by specific kinases. ATP transfers adenylyl (AMP) group in the activation of substrates such as amino acids in the first step of protein synthesis, which is the formation of aminoacyl adenylate. This is one of the ways in which ATP can act as intermediate in biochemical reactions. ATP also acts intracellularly in the regulation of the activities of proteins by binding to their regulatory subunit and acting as an allosteric effector. Extracellularly, by interacting with their specific receptors, ATP and adenosine initiate either a metabolic signal or an ion channel signal, which then bring about initiation of specific cellular activities.
Passwater: How is ATP made in the body?
Rapaport: Intracellular ATP is present at high concentrations, from 2.2 millimolar in red blood cells to 3 millimolar in liver cells to 2-10 millimolar in skeletal muscle cells.
There are three main processes for extracting energy from the food we eat and storing this energy in ATP. The lesser pathway if called glycolysis, which is the first group of reactions in converting carbohydrates into cellular ATP. It is carried out in the cytosol and does not require oxygen presence. It is the breakdown of the sugar glucose, either from molecular glucose itself or from glycogen, which is a cellular storage form for glucose, to two three-carbon units, producing ATP molecules from ADP in an inefficient process that proceeds anaerobically. Thus the breakdown of glucose, which is termed glycolysis (Figure 2), although energetically inefficient, is highly desirable since it is not dependent on oxygen presence and can therefore take place under poor blood flow conditions. Hence the old adage “sugar is good for the heart”.
Figure 2: Production of ATP by glycolysis.
Passwater: So the net production of ATP from glycolysis is only two molecules of ATP for each molecule of glucose. There are four molecules of ATP produced but two molecules are used in the reactions which leaves a net production for two molecules of ATP after ten enzymatic reactions involved in glycolysis.
What is the second way ATP is produced in the body from metabolism?
Rapaport: A second mode of ATP synthesis is from pyruvate in the citric acid cycle (Figure 3). Pyruvate is a major three carbon unit product of glucose catabolism and by the catalytic action of pyruvate dehydrogenase yields acetyl coenzyme A. Acetyl coenzyme A is then utilized for ATP synthesis in the mitochondria in a sequence that is also dependent on oxygen presence but to a lesser extent than the oxidation of fatty acids.
Figure 3: The production of ATP by the citric acid cycle. The reduced forms of NAD+ (NADP+) and FAD (NADH and FADH2 respectively) are reoxidized by the electron transport system in the step producing ATP from ADP.
Passwater: Well this is more productive than glycolysis as 36 molecules of ATP are produced from a molecule of glucose via the citric acid cycle and oxidative phosphorylation. What’s the most efficient way of producing ATP?
Rapaport: However, the most effective mode of ATP synthesis by phosphorylation of ADP, is by beta oxidation of fatty acids in the mitochondria, which is a cellular organelle where aerobic metabolism takes place. Beta oxidation of fatty acids is energetically a very effective pathway, however, it is totally dependent on oxygen presence and therefore extremely sensitive to blood flow.
Passwater: For our non-chemist readers, beta oxidation is simply the snipping off of a two-carbon unit from the backbone of a fatty acid at the second carbon called the “beta” carbon in from the carbonyl end group. This is a four-step process. Each cleavage of two carbons results in the production of one molecule each of NADH, FADH2 and Acetyl-CoA. Each molecule of NADH leads to producing three molecules of ATP. Each molecule of FADH2 produces two molecules of ATP and each molecule of Acetyl-CoA eventually produces 12 molecules of ATP. So when a molecule of a fat such as the 16-carbon Palmitate is oxidized it leads to the formation of 129 molecules of ATP. [(7x3) + (7x2) + (8x12) = 131 – 2]
Thus a typical fatty acid such as palmitate yields 129 molecules of ATP, whereas the sugar glucose yields only 38 molecules of ATP, 2 from glycolysis and 36 from the citric acid cycle and oxidative phosphorylation.
Do we make enough ATP for our needs?
Rapaport: Only when we are young and do not suffer from stress or ailments. Reactive oxygen species (ROS), generated constantly near the mitochondrial membrane act in producing mutations in mitochondrial proteins and within time, significantly decrease mitochondrial function (aerobic ATP synthesis, respiration or oxidative phosphorylation). This is what happens upon aging whereby skeletal muscle mitochondrial ATP synthesis drops to half of what it is in adults. Other organs are also similarly affected leading to a drop of 50% in total blood (red blood cell) ATP levels. Hence the efficacy of antioxidants that Dr. Passwater has been touting for so long. By capturing the reactive oxygen species or intermediates, antioxidants support cellular energetics.
Passwater: Could health be improved if we could increase our ATP levels?
Rapaport: Under normal circumstances, yes and in a large variety of different mechanisms.
Passwater: Well that covers most of the fundamentals. Let’s take a break and pick up again next month. WF
© 2006 Whole Foods Magazine and Richard A. Passwater, Ph.D.
This article is copyrighted and may not be re-produced in any form (including electronic) without the written permission of the copyright owners.