Spin Doctor

In 1956 I was in Oxford beginning research for a DPhil in Sir Hans Krebs’ Medical Research Council Unit on the structure and function of isolated mitochondria. This involved smashing open cells in a solution of sucrose with a spinning pestle and mortar, followed by more spinning in a differential centrifuge to isolate particles according to their size. The cell nuclei were sedimated first followed by the mitochondria. We now understand that mitochondria are tiny organelles that are often referred to as the energy powerhouses of cells or our body's batteries. Their prime function is to take in the sugars that have been converted from the food we eat, and turn them into a form of energy that cells can use. Each mitochondrion spans 16 kilobases of DNA encoding 37 genes. But when I began my D.Phil programme there was actually a lively debate as to whether mitochondria were really artefacts of processing cells for histological examination. This entailed examining the isolated mitochondria with an electron microscope, then newly installed in Oxford's department of anatomy and comparing their structure with that of intact cells in organs.

Three years later these doubts as to whether mitochondria were an authentic part normal cells had been overcome. My part in this saga was that I had isolated suspensions of mitochondria in sucrose solutions as distinct functional units with a perspective on their evolution that spanned organisms right across the animal kingdom. My doctorate work demonstrated that these suspensions of organelles retained the membrane structure that was their histological characteristic, and also without exception contained the enzymes of the Krebs cycle. The Krebs cycle, also known as the citric acid cycle, is a sequence of enzyme-catalyzed reactions in living cells that is the final series of reactions of aerobic metabolism of carbohydrates, proteins, and fatty acids, and by which carbon dioxide is produced, oxygen is reduced, and ATP, the energy currency of cells, is formed. My papers were published in the Journal of Biochemistry before I received my doctorate. A new discovery was that they converted glutamic acid to aspartic acid, a previously unknown process. This conversion turned out to be a neat proof of the central role played by the citric acid cycle as the carbon carrier of intermediary metabolism in mitochondria. Incidental outcomes of my post graduate research were the discovery of acetylcholine-containing vesicles in rat brain, and DNA in mitochondria. The former was an important piece of work which opened up the mechanism by which nerve impulses passed between cells. The latter discovery I am sorry to say I dismissed as contamination from ruptured nuclei ! It has since turned out to be a key piece of evidence for evolution of cells in plants and animals being brought about by infection with a free-living microorganism (the mitochondrion) which subsequently was unable to live outside the cells of its host.

Krebs, as the discoverer of the citric acid cycle, was then at the centre of the British/US biochemical universe and during my six student years all the main principles of the subject were established and integrated; glycolysis, oxidative metabolism, protein synthesis, carbon fixation and the alpha helix replication of DNA. These discoveries were delineated by a steady succession of researchers from the four corners of the globe who visited Krebs' lab where I worked, .

I then began postdoctoral work in Oxford with a one-year appointment as a departmental demonstrator, running Kreb’s practical classes on manometry for medical students. The idea was to help him produce a book on this technique, which had served him well in his Nobel Prize work. But we soon concluded that manometry, as a key investigative tool of intermediary metabolism, was no longer its driving force. In fact, for my own research on the small amounts of mitochondria isolated from invertebrates, such as flies, I had to make a version of the newly invented oxygen electrode to measure the uptake of oxygen by these small amounts of mitochondrial material. The book on manometry was abandoned.

Regarding a future research path, I reasoned that since all the major principles of cellular energy production had been established, to stick with this field would lead me into wars of attrition filling in the minutia behind the big picture. It seemed to me that the next major problem was at a higher chemical order and concerned the problem of how cells are able to communicate within and between organs. Fortuitously, the previous year I had been offered a post-doc fellowship with Ian Chester Jones, who had recently been appointed to the chair of zoology in Sheffield. His team was studying the actions of hormones of the adrenal cortex. My task was to find suitable vertebrate models for a comparative biochemical study of the role of these steroids in salt and water movement across cell membranes. I chose eels for this work because of the vital switch in the direction of sodium transport during their migration between seas and rivers. In particular, they expelled sodium on their migration from their birthplace in the Sargasso Sea and took it up on entering the freshwater of European rivers. My interest in this topic actually began when, as a biochemistry undergraduate, I came across Baldwin’s Comparative Aspects of Biochemistry. I was particularly intrigued by the idea that the first ancient cells had formed in a primeval sea poor in sodium and rich in potassium. This idea was bound up with the question as to why the mitochondria of all modern organisms have to be bathed in potassium salts rather than sodium. This is still a major problem of animal evolution and it really set the scene for me to move from biochemistry into zoology. Actually, my paper on Myxine, the primitive marine hagfish, with a blood system and physiology that predated that of the true fishes, was connected to this early fascination with the evolution of electrolyte compartmentation (CPB Volume 3 Issue 3 October 1961 Pages 175-183). This paper was also chosen for the celebratory 2008 edition of Comparative Biochemistry and Physiology .

The isolated eel gill arches I used in Oxford were to be the first step towards establishing suspensions of their salt transport cells as a prelude to the isolation of the cellular components of sodium pumps in cell walls which maintain an intracellular salt composition as a dynamic equilibrium. Advice was on hand in Oxford, from Ron Witham, who a few doors away was pioneering the study of sodium transport in red cells, and Walter Bartley in Kreb’s Medical Research Council Unit who was working on sodium and potassium regulation in isolated mitochondria. It is interesting that this line of enquiry at the cellular level in eel gills was not revisited by anyone until Peter Pärt’s work in 2002.