Miracle Man of Miracle Drugs
Dr. Yellapragada Subbarow

 
ATP - Energy for Life
 

One of the observations Dr SubbaRow made while testing the phosphorus method seemed to provide a clue to the mystery what happens to blood sugar when insulin is administered. Biochemists began investigating the problem when Frederick Banting showed that injections of insulin, the pancreatic hormone, keeps blood sugar under control and keeps diabetics alive.

SubbaRow worked for 18 months on the problem, often dieting and starving along with animals used in experiments. But the initial observations were finally shown to be neither significant nor unique and the project had to be scrapped in September 1926.

Out of the ashes of this project however arose another project that provided the key to the ancient mystery of muscular contraction. Living organisms resist degeneration and destruction with the help of muscles, and biochemists had long believed that a hypothetical inogen provided the energy required for the flexing of muscles at work.

Two researchers at Cambridge University in United Kingdom confirmed that lactic acid is formed when muscles contract and Otto Meyerhof of Germany showed that this lactic acid is a breakdown product of glycogen, the animal starch stored all over the body, particularly in liver, kidneys and muscles. When Professor Archibald Hill of the University College of London demonstrated that conversion of glycogen to lactic acid partly accounts for heat produced during muscle contraction everybody assumed that glycogen was the inogen. And, the 1922 Nobel Prize for medicine and physiology was divided between Hill and Meyerhof.

But how is glycogen converted to lactic acid? Embden, another German biochemist, advanced the hypothesis that blood sugar and phosphorus combine to form a hexose phosphoric ester which breaks down glycogen in the muscle to lactic acid.

In the midst of the insulin experiments, it occurred to Fiske and SubbaRow that Embden's hypothesis would be supported if normal persons were found to have more hexose phosphate in their muscle and liver than diabetics. For diabetes is the failure of the body to use sugar. There would be little reaction between sugar and phosphorus in a diabetic body. If Embden was right, hexose (sugar) phosphate level in the muscle and liver of diabetic animals should rise when insulin is injected.

Fiske and SubbaRow rendered some animals diabetic by removing their pancreas in the spring of 1926, but they could not record any rise in the organic phosphorus content of muscles or livers after insulin was administered to the animals. Sugar phosphates were indeed produced in their animals but they were converted so quickly by enzymes to lactic acid that Fiske and SubbaRow could not detect them with methods then available. This was fortunate for science because, in their mistaken belief that Embden was wrong, they began that summer an extensive study of organic phosphorus compounds in the muscle “to repudiate Meyerhof completely”.

The departmental budget was so poor that SubbaRow often waited on the back streets of Harvard Medical School at night to capture cats he needed for the experiments.

When he prepared the cat muscles for estimating their phosphorus content, SubbaRow found he could not get a constant reading in the colorimeter. The intensity of the blue colour went on rising for thirty minutes. Was there something in muscle which delayed the colour reaction? If yes, the time for full colour development should increase with the increase in the quantity of the sample. But the delay was not greater when the sample was 10 c.c. instead of 5 c.c. The only other possibility was that muscle had an organic compound which liberated phosphorus as the reaction in the colorimeter proceeded. This indeed was the case, it turned out. It took a whole year.

The mysterious colour delaying substance was a compound of phosphoric acid and creatine and was named Phosphocreatine. It accounted for two-thirds of the phosphorus in the resting muscle. When they put muscle to work by electric stimulation, the Phosphocreatine level fell and the inorganic phosphorus level rose correspondingly. It completely disappeared when they cut off the blood supply and drove the muscle to the point of "fatigue" by continued electric stimulation. And, presto! It reappeared when the fatigued muscle was allowed a period of rest.

Phosphocreatine created a stir among the scientists present when Fiske unveiled it before the American Society of Biological Chemists at Rochester in April 1927. The Journal of American Medical Association hailed the discovery in an editorial. The Rockefeller Foundation awarded a fellowship that helped SubbaRow to live comfortably for the first time since his arrival in the United States. All of Harvard Medical School was caught up with an enthusiasm that would be a life-time memory for con­temporary students. The students were in awe of the medium-sized, slightly stoop shouldered, "coloured" man regarded as one of the School's top research workers.

SubbaRow's carefully conducted series of experiments disproved Meyerhof’s assumptions about the glycogen-lactic acid cycle. His calculations fully accounted for the heat output during muscle contraction. Hill had not been able to fully account for this in terms of Meyerhof's theory. Clearly the Nobel Committee was in haste in awarding the 1922 physiology prize, b­ut the biochemistry orthodoxy led by Meyerhof and Hill themselves was not too eager to give up their belief in glycogen as the prime source of muscular energy.

Fiske and SubbaRow were fully upheld and the Meyerhof-Hill­ theory finally rejected in 1930 when a Danish physiologist showed that muscles can work to exhaustion without the aid of glycogen or the stimulation of lactic acid.

Fiske and SubbaRow had meanwhile followed a substance that was formed by the combination of phosphorus, liberated from Phosphocreatine, with an unidentified compound in muscle. SubbaRow isolated it and identified it as a chemical in which adenylic acid was linked to two extra molecules of phosphoric acid. By the time he completed the work to the satisfaction of Fiske, it was August 1929 when Harvard Medical School played host to the 13th International Physiological Congress. ATP was presented to the gathered scientists before the Congress ended. To the dismay of Fiske and SubbaRow, a few days later arrived in Boston a German science journal, published 16 days before the Congress opened. It carried a letter from Karl Lohmann of Meyerhof's laboratory, saying he had isolated from muscle a compound of adenylic acid linked to two molecules of phosphoric acid!

While Archibald Hill never adjusted himself to the idea that the basis of his Nobel Prize work had been demolished, Otto Meyerhof and his associates had seen the importance of Phosphocreatine discovery and plunged themselves into follow-up studies in competition with Fiske and SubbaRow. Two associates of Hill had in fact stumbled upon Phosphocreatine about the same time as Fiske and SubbaRow but their loyalty to Meyerhof-Hill theory acted as blinkers and their hasty and premature publications reveal their confusion about both the nature and significance of Phosphocreatine.

The discovery of ATP and its significance helped reveal the full story of muscular contraction: Glycogen arriving in muscle gets converted into lactic acid which is siphoned off to liver for re-synthesis of glycogen. This cycle yields three molecules of ATP and is important in delivering usable food energy to the muscle. Glycolysis or break up of glycogen is relatively slow in getting started and in any case muscle can retain ATP only in small quantities. In the interval between the begin­ning of muscle activity and the arrival of fresh ATP from glycolysis, ­Phosphocreatine maintains ATP supply by re-synthesizing it as fast as its energy terminals are used up by muscle for its activity.

Muscular contraction made possible by ATP helps us not only to move our limbs and lift weights but keeps us alive. The heart is after all a muscle pouch and millions of muscle cells embedded in the walls of arteries keep the life-sustaining blood pumped by the heart coursing through body organs. ATP even helps get new life started by powering the sperm's motion toward the egg as well as the spectacular transformation of the fertilized egg in the womb.

Archibald Hill for long denied any role for ATP in muscle contraction, saying ATP has not been shown to break down in the intact muscle. This objection was also met in 1962 when University of Pennsylvania scientists showed that muscles can contract and relax normally even when glycogen and Phosphocreatine are kept under check with an inhibitor.