 As techniques of medical practice advance, the doctor relies more and more on knowledge gained through laboratory studies. The clinic and the biochemical laboratory are merging forces in a joint attack upon disease. The common ground of the present attack is inside the cell. In every cell, metabolism, chemical change, goes on without stop as long as life persists. The biochemists are determining pathways, establishing an order and a pattern of the chemical life processes. Glucose metabolism, one of the best understood of these processes. Each chemical change within a cell is catalyzed by a specific enzyme. The rate of change may be the determining factor between health and disease. Sometimes an enzyme works smoothly and well, sometimes hesitantly, and other times not at all. What are the factors regulating enzyme functions? Knowing the answer may help us to control enzyme action and so directly attack disease at its source inside the cell. But in actual living cell, we see little of the constant chemical change, the building up, the breaking down, the action which must be studied in the quest for knowledge of enzymes and metabolism. Here are the bare essentials of the biologist's concept of cell structure. The cell membrane, within it the nucleus, mitochondria, microzones, and other granules inside the cytoplasmic fluid. In the search for the chemical facts of life, the biochemists must visualize the invisible enzyme. What factors make an enzyme function or cease to function? First, the location of the enzyme within the cell. A change of location may bring on a change of action. Second, enzyme quantities. More enzymes, more action. More chemical change takes place. Third, the enzyme's chemical environment influences its action. When the chemical surroundings change, there is a change in the amount of work done. These factors influencing enzyme action are the subject of our studies. Laboratory methods of locating the place of enzyme action have been worked out. In one of these, homogenization of the tissue cells is the first step. Rat liver homogenate in sucrose solution is prepared by Dr. W. C. Schneider. He first checks the homogenate to make sure that the cells have been well broken up. Only an occasional cell escapes destruction. The nuclei and cytoplasmic granules of the disrupted cells are dispersed in the solution. Then, through a series of steps, the centrifugal force separates the homogenate into its component parts. The homogenate begins to separate into parts representing specific regions of an individual cell. The supernatant fluid is centrifuged again. Equal amounts of the homogenate and all four fractions representing parts or regions of the cell are prepared to be tested for enzyme activity as measured by oxygen uptake. Dr. Schneider finds that almost all of the succinic dehydrogenase action is localized in class 3, containing the mitochondria. Inside the cell, the succinic dehydrogenase functions here. The localization of enzyme action is studied with different approaches by other scientists. Working at the Marine Biological Laboratory at Woods Hole, Massachusetts, Nobel Prize winner St. Georgie demonstrates the effect of adenosine triphosphate ATP on a muscle fiber. A series of chemical steps over a period of weeks has reduced the muscle to a state bearing little resemblance to its appearance in life. This might be called a demonstration of vital activity with dead cells and chemicals. Under these conditions, when ATP is applied to the fiber, contraction takes place. A dead fiber contracts as if alive. Striving towards smaller fields and higher magnifications, the scientist breaks down the muscle fiber still further to obtain liquid actomyosin, the contractile protein of muscle. This liquid is formed in threads. Threads, which are not muscle, yet can be made to act like muscle under the influence of ATP. When contraction takes place, the important enzyme, ATP-8, functions. This releases inorganic phosphate and energy which becomes available for muscle relaxation. The blue color indicates the presence of inorganic phosphate. This ATP-8 activity sets in when the cells are fully contracted, probably as a result of the change of enzyme location brought on by the change shape of the cell. In other cells, too, a changed state of activity may bring about a change of enzyme location which affects enzyme action. Fertilization alters the cell structure. Molecules formerly isolated now come into contact. In the sea urchin egg, this change in the state of activity leads to a change of enzyme location. The result is a marked increase in enzyme activity of the cytochrome system. When a muscle contracts, its cells change shape. Thus, the complex interrelation between enzyme location and the state of activity of the cell is an important factor influencing enzyme action. The quantity of enzyme also may be the determining factor in chemical changes taking place in a cell. That enzyme amount changes at different times is strikingly illustrated by the work of doctors Meyer and McChan on the succinic dehydrogenase content of the corporelutia of rat ovaries. For the first few days of the three-week pregnancy, there is little change in enzyme concentration. Then, the enzyme concentration rises rapidly, reaching its maximum at the 11th day. This enzyme quantity per unit weight, though more than twice that at conception, is maintained throughout the rest of pregnancy, even while the total gland tissue is increasing some 300% in weight. Then, at parturition, the enzyme concentration drops sharply. Four days later, it has returned to its original value. Different states of body activity are accompanied by a change of enzyme quantity. This change in succinic dehydrogenase concentration may be correlated with hormonal changes occurring during pregnancy. Hormones are part of the enzyme's chemical environment. This environment may vary in a great many ways, and each variation affects enzyme action. That hormone, an important element of enzyme environment, can affect the enzyme quantity of tissue is indicated by the work of Dr. Samuel Tipton. This is glucose metabolism when the hormones are normally balanced. Increase of thyroid activity upsets the balance and markedly increases the activity of these enzymes. Cytochromoxidase and succinic dehydrogenase. A reduction of the amount of thyroxin decreases the quantity of these enzymes and restores the balance. Another aspect of chemical environment is pH, relative acidity or alkalinity. Each enzyme works best at a specific pH. One enzyme may function only in an environment with this degree of acidity. Action may slow, then cease as the environment becomes more acid. The same thing may happen when the balance is tipped to the alkaline side. Another enzyme may work well only at a quite different pH. pH changes which affect enzyme activity may in turn result from enzyme action. For example, lactic acid produced by enzyme action in glycolysis increases the acidity of a cell. So a product of metabolism becomes a factor regulating enzyme action. Similarly, the inorganic phosphate which is produced in these steps of metabolism acts as a regulator in other steps. These other steps where phosphate is used as substrate are regulated by the inorganic phosphate produced. Another established point of vulnerability of enzyme action is the coenzyme which may also be attacked through its chemical environment. Coenzyme formation may involve the combination of vitamin and phosphate groups. This coenzyme formation is also the result of enzyme action. Antivitamins and other anti-metabolites have been shown to slow enzyme action apparently by stopping coenzyme formation. Research has named other factors of environment which influence enzyme action. Normally in laboratory experiments when enzymes and substrate are brought together under carefully controlled conditions there is a reaction. Sometimes in similar experiments the mixing of substrate and enzyme brings no reaction. An activator may be missing from the chemical environment. For example, enolase, one of the essential enzymes of glycolysis, functions only when magnesium is present as an activator. Experiments identifying special activators of enzymes are significant steps toward a rational chemotherapy. Knowledge of enzyme inhibitors is equally significant. Though the protein of enzymes is of great complexity, some details of the molecular structure of some enzymes are known. FH groupings are essential to the function of many of these. Blocking the FH inhibits the enzymes. For instance, arsenic of the vesicant wargath lewisite ties up the FH group and inhibits the enzyme. That is when the arsenic is present the substrate cannot join with the protein of the enzymes. BAL acts to prevent injury by competitively attracting the arsenic which blocks the FH of the enzymes. Once again the molecular action. Lewisite gas attacks through the enzymes chemical environment. BAL provides protection through the chemical environment. This protection was made possible because of fundamental knowledge of factors regulating enzyme action. Other drugs long in use owe their action to effects on important cell enzymes. Eseren specifically inhibits an important enzyme of nerve tissue, acetylcholine esterase, a cross section showing the active portion of the nerve fiber. Potassium ions are in abundance inside and sodium in abundance outside. This is the electrical fixture, plus outside minus inside. Dr. David Nachman's son of Columbia University attributes an important role in conduction to this enzyme which functions inside the active surface of the nerve. The electrical fixture is the same throughout the length of the nerve. When a stimulus occurs, a flow of current is initiated. This releases acetylcholine. The electrical resistance of the membrane lessens and ions interchange more freely. Polarity reverses. Acetylcholine esterase now enters the fixture in the active portion of the nerve. The current flows in the next unit and more acetylcholine is released and ions move again. Meanwhile, the enzyme has acted on the acetylcholine and restored the first unit of the nerve to its resting state. Prepared it for the next impulse and so the impulse passes down the nerve. The nerve impulse is a complex interrelation between electrical and chemical factors in which enzymes play an important part. Ethyrins, by inhibiting acetylcholine esterase, blocks the nerve impulse. In demonstrating the effect of ethyrins, Dr. Nachman finds an ideal subject in the common squid. The giant axon of the squid is a single nerve fiber, the largest single nerve fiber known to exist. The nerve fiber is quickly mounted and while still conductive is placed in an electrical circuit so that it can be stimulated and so that the nerve impulses resulting from the stimulation can be seen and measured on the oscilloscope screen. The machine which measures my new electrical impulses of the magnitude produced by nerve is adjusted. This is the pattern of the nerve potential as it is rhythmically stimulated. Ethyrin inhibits acetylcholine esterase. As the nerve is exposed to ethyrins, it rapidly loses its ability to transmit impulses. Then the supply of ethyrin is cut off and a flushing bath of seawater is released. As the ethyrin is washed away, the enzyme inhibition is released. The nerve rapidly recovers its ability to transmit the impulse. Ethyrin and other drugs such as strychnin, DFP and prokane because of their molecular structure can penetrate the structural barrier of a nerve fiber and acts directly on the conductive membrane at any point of the nerve surface as well as upon the synapse. Other drugs such as curarin, acetylcholine and prosigmens which cannot penetrate the structural barrier are thought to act on the postsynaptic membrane. Frequently, the biochemist works not with living cells, but with isolated enzyme systems from disrupted cells. His charts are drawn from in vitro findings. Are they true charts of in vivo conditions? Tracer studies with living normal animals help give the answer. An experiment is presented as adapted for the medical biochemistry course at Western Reserve University Medical School. More exact methods would be used in research. Food containing radioactive carbon atoms has been used by Dr. Harland Wood and Dr. Victor Lorber to put in vitro experiments to the test in the intact animal. In order to determine the radioactivity of the food, carboxyl-labeled acetate, it must be spread in a thin film. It is placed directly under the window of the Geiger countertube. You will note that it takes 15 seconds for a standard number of counts. In this case, 2048, to be recorded by a given amount of material. The acetate, with its radioactive label, enters the path of metabolism here. The C14 acetate is fed to the living test subject. The animal is placed in a closed jar. Its respiratory CO2 is swept into cubes containing alkali by a brisk stream of CO2-free air. The experiment is terminated after three hours and the distribution of C14 is studied. The alkali containing respiratory CO2 is assayed for radioactivity to determine the extent of oxidation of the acetate. Barium carbonate is prepared as a thin layer on a filter paper dip. This is dried and, after weighing, is placed on the brass holder for counting. It can be calculated from these radioactivity measurements that 40% of the acetate ends here. In addition, the metabolism of the acetate is traced in the animal using liver glycogen as an indicator of events. The rat is anesthetized. The liver is removed and placed in a tube containing strong alkali and the process of isolating the glycogen is begun. If the Krebs cycle and glycolysis have occurred in the animal as on the biochemist chart, then labeled carbon can end up in the glycogen of the rat's liver even without a net increase in quantity of glycogen. But the tagged carbon can occupy only certain positions in the glucose molecule. The C14 can be followed as the acetate and unlabeled oxaloacetate condense, performing citrate. Then disconnectate, which in turn forms isocitrate. Then oxalo succinate, alpha ketoglutarate, and succinate. Succinate is the first symmetrical molecule we encounter. It changes to fumarate, which is still symmetrical. Now hydration occurs. The OH will attach in this position in half the molecule. In the other half, it will attach here. Now we have two forms of unsymmetrical malate. When one is reversed, we see that it differs from the other only in the position of the isotope. These two differently labeled malates form correspondingly different oxaloacetate. Now by beta decarboxylation, the oxaloacetate produce peruvate. One labeled the other not. By reversal of glycolysis, two three carbon molecules are put together, and the glucose units of glycogen are formed. Numbers indicate carbon positions in the glucose molecule. The labeled carbon is in position three, by a different combination of the same peruvate, two other glucose molecules form. But only the third and fourth carbon can be radioactive if the biochemist's chart is right. Continuing the experiment, the liver glycogen is isolated preparatory to chemical degradation. Here we see the glycogen being precipitated with alcohol. The glycogen is hydrolyzed to glucose, which is then fermented by bacteria to lactic acid. The lactic acid is then oxidized with permanganate to yield the carbon in two fractions. Carbon three and four appearing as carbon dioxide, and carbon one, six, two, and five as acetaldehyde. Here the permanganate has been added, and the volatile acetaldehyde precipitates as the yellow 2,4 dinitrophenol hydrazone, and the CO2 as barium carbonate. These precipitates are prepared for counting by plating in the usual manner. As the chart drawn from in vitro experiments indicated, the in vivo demonstration shows the carbon to be in positions three and four only. With the carbon from positions three and four, the time required for 2048 counts is six and a half minutes. Whereas with the carbon from positions one, two, five, and six, 60 minutes is required, practically a background reading. When in similar experiments, methyl-labeled acetate is fed to the animal, the radioactivity of the three and four carbon atom fractions, and that of the one, two, five, and six carbon atom fraction is the same. Both fractions require almost the same time for counting. Thus, the carbon 14 shows up in all six positions of the carbon ring of glucose. This strongly validates the chart. Such experiments make us confident that the picture of glycolysis and the Krebs cycle drawn by biochemists is in general the true picture of chemical changes inside the cells of the living animal. Bacteria, like all living cells, contain enzymes. The life catalysts of the bacteria are as vulnerable as those of our own cells. The energy-releasing pattern of bacterial metabolism is similar to our own. To kill off an invading organism, an attack could be planned against a vital but weak enzyme of the bacteria. If the enzyme happened to be strong in the host and weak in the invader, the chemical attack would be effective. This is the basis of the action of the sulfur drug. Sulfonylamide is very much like para-amino-benzoic acid, differing only in the last few atoms. P-A-B-A is an integral part of the B vitamin, folic acid. Folic acid is essential to bacteria and will probably be shown to be part of a coenzyme. The sulfur molecule is similar enough in structure to compete successfully with the P-A-B-A for the active center of the enzyme. However, not being identical, the complete action cannot take place. So the united structure of folic acid is not formed and a coenzyme cannot form. Again, the action of the sulfur drug. No longer able to divide with a non-functioning coenzyme of an essential enzyme, the bacteria are no match for the disease-fighting mechanisms of the host. Dr. D. W. Woolley of the Rockefeller Institute for Medical Research shows the effects of an anti-metabolite on enzymes of mice. Mouth 1 is on a normal diet. Mouth 2 has been fed a normal diet with adequate thiamine, but in addition, this mouse has received the anti-metabolite pyrethiamine. The typical thiamine deficiency symptoms are visible. Here is the story chemically. This is the formula of thiamine. If the sulfur atom is replaced by two carbons and hydrogen, thiamine becomes pyrethiamine. Similar, but not identical molecules. An enzyme which is specific for thiamine accepts the molecule as substrate. Two phosphate molecules are added. A coenzyme essential in glucose metabolism is formed. This coenzyme is now free to join with the protein to form oxidative decarboxylase and function in metabolism. Since the structure of pyrethiamine is so very like thiamine, it too can join with the enzyme which is specific for thiamine. However, the slight difference in structure does not allow the chemical action. No coenzyme is formed. In this way, pyrethiamine competitively inhibits the formation of an essential coenzyme. Again, the mechanism of anti-metabolite action. Without the coenzyme, there is no oxidative decarboxylase. Glucose metabolism is therefore inhibited at these steps. Many other anti-metabolites are already known. Is it possible to affect specifically the abnormal cells of the body? If one enzyme were more important to the abnormal cell than to the normal, that would be the enzyme to inhibit by an anti-metabolite. For example, glycolysis is apparently more important to cancer cells than to normal cells. Perhaps a molecule can be fashioned capable of inhibiting the decisive energy-yielding step of glycolysis. The proper modification of the coenzyme might yield the chemical weapon to kill the cancer cell. The growing knowledge of enzyme regulation and especially of anti-metabolite holds promise of a more advanced chemotherapy. A rational fashioning of a specific chemical weapon for each disease. Such specific agents have long been the dream of medical men. It is the hope of men and women who follow the chemical paths inside the cell that their work may help fashion these weapons which are so essential in mankind's continuing fight against disease.