Monitoring moving molecules

Emeritus Professor Philip Kuchel reflects on the moving spectrum of a distinguished career in biochemical research.

Lab+Life Scientist: What drew you to become a biochemist?

Professor Philip Kuchel: The idea that you could explain life processes with biochemistry was, and still is, really fascinating to me - for example, a defect in one enzyme will produce a multisystem disorder in a patient.

I was lucky enough to be admitted into medical school at Adelaide University and by the end of first year I had decided I would pursue medical research in some form; and in second year I took a real liking to biochemistry.

Although there was a lot of memory work in the medical subjects, the challenge served a valuable purpose as it forced me to develop strategies for learning - I basically ‘learnt how to learn’ in second-year medicine.

I treated a biochemical pathway like some of the word games that involve transformation of a word from one to the next. And I got the bigger picture. I was able to remember structures sufficiently well to get an overview of whole metabolic systems - once you do that it all falls into place.

I took a year off the main course after the third-year examinations and worked with Professor Bill Elliot and Dr George Rogers; I was given the run of the lab.

I had access to sophisticated instruments like the electron microscope, which was a big deal back in those days. I could even run it myself! Here I was, a mere honours student, and they trusted me with expensive instruments.

I think that’s one of the things that drew me in - for the first time I had senior people around me who were enthusiastic about what I was discovering.

L+LS: So instead of pursuing medicine you took on a PhD?

PK: By the end of my bachelor of medical science I went into fourth-year medicine, but by that stage I had decided I would pursue biomedical science alongside clinical medicine. I enrolled in additional bachelor of science courses in pure maths and computing during the next three years because I wanted to hone my analytical skills. Then I did my residency on the academic medical and surgical units at Royal Adelaide Hospital aiming to see how to combine research and clinical work.

A big turning point was when I made the choice to enrol in a PhD in physical biochemistry with Professor Laurie Nichol and Dr Peter Jeffrey in the JCSMR at the Australian National University (ANU). Amongst other things the work introduced me to computer modelling of enzyme kinetics using new numerical methods of solving otherwise intractable differential equations.

That was a higher order development for me - it was one thing to have a map of the chemicals in a metabolic pathway, the next thing was to measure how fast they were going and predict the rate of a reaction pathway.

I was sharing a lab with postdoc Dr Dave Roberts, who arrived from Belfast and had been commissioned by Cambridge University Press to write a textbook on enzyme kinetics. He asked me to refine some of the mathematical derivations in it - that was quite a buzz for me as a PhD student.

We developed a computer model of the human urea cycle that could predict the time course of metabolic events. Dave used the Univac 1108 mainframe computer at ANU and it took over the whole core memory of 128 K to simulate 10 minutes of metabolism in 10 minutes!

We were making incredible assumptions about the distribution of the enzymes inside the cell, but what we calculated were metabolite concentrations that matched pretty well with what was reported in the clinical biochemical literature.

We could actually predict the levels of the metabolites that would arise when one of the enzymes was defective.

That was when I realised that by doing physical chemistry you could predict a clinical outcome. That was really exciting.

L+LS: Did you consolidate your career as a research academic at the University of Sydney?

PK: Yes, most definitely! The University of Sydney was looking for someone to foster links between biochemistry and the Faculty of Medicine - biochemistry remains in the Faculty of Science to this day, but a lot of the inspiration for what we do in research, and what we teach, comes from medicine.

I was fortunate to have excellent funding right form the start of my career. I had two 5-year NHMRC project grants in succession. The first one I received was in the early 1980s and that really set me up.

I moved to the University of Sydney in 1980 and helped reshape the Biochemistry Department, working very closely with Professor Gerry Wake until the end of 1999 when he retired. Up until 1995 we alternated chairmanship of the department in 2-year stints. That suited both of us really well as we had periods of respite without losing touch with administrative and academic events that were important to the research and educational success of the department. We had the same view on research and teaching - teaching was to be research inspired, not just storytelling, and it was to be problem-solving.

I recall Gerry saying to me, “If your research is going well you can handle anything else (such as administration and teaching)”; that was also my view.

Having continuity, in infrastructure and the people around you, and a sense of security in your job is important for a research career.

Another important person throughout my career was Dr Bob Chapman, whom I had employed with my first grant at the University of Sydney. We worked together almost our whole careers.

Bob could make the NMR spectrometers ‘sing’. He could operate them for hours on end; consequently, the research students were great beneficiaries of his ever-presence and wealth of experience. He was a no-nonsense individual, so if you could get an idea or interpretation of some NMR data past Bob you had done well. This was excellent training for the students.

L+LS: How has access to infrastructure dictated your research choices?

PK: This has been vital in our particular line of research.

When we finally mustered enough money to buy our own NMR spectrometer, I decided on a wide-bore instrument with a view to adding extra magnetic coils inside it so we could measure molecular diffusion. I was not sure where this would lead, but I knew that the diffusion coefficient of a molecule is a fundamental property that could reflect how it takes to encounter an enzyme that operates on it.

Furthermore, how neat would it be to measure how fast molecules move around inside cells!

This was linked to our curiosity about the timescale of events in living systems - how long does it take for a molecule to move from one side of a cell to the other? Surely that transit time determines in a very fundamental way why cells are the size they are.

Ours was the first super-conducting magnet in the world with magnetic-field-gradient coils in it. We could measure diffusion of small molecules in red blood cells and, perhaps more interestingly, we measured the rate of diffusion of haemoglobin in red blood cells. That became a benchmark experiment for us.

We had reasoned that if we developed the technology to a point where we could measure how fast haemoglobin was moving around inside the cell then that would open up a raft of other experiments based on measuring diffusion. And, indeed, it turned out that way. Magnetic field gradients have become a central part of modern NMR spectroscopy, and pulse sequence development.

L+LS: How do you think NMR compares to other methods for measuring the chemical basis of life?

PK: That is a leading question! So I will be somewhat ‘tongue in cheek’ and suggest that in the quantitative (measurement) sciences ‘there is spectroscopy and there are other things - like gas chromatography and mass spectrometry’. And ‘in spectroscopy there is NMR spectroscopy and there are the rest!’ With respect to scope and adaptability to novel contexts across all of science, NMR ‘has it in spades’!

I think the reason for this is that chemistry is all about the electrons around atomic nuclei. The electrons are the basis for connecting other atoms to build up molecules. The electron cloud around each atomic nucleus influences its local magnetic field so when you change chemistry you change the electron cloud and you change the NMR resonance (absorption) frequency.

NMR is exceedingly sensitive to what happens to that electron cloud and no other spectroscopy comes near it with respect to versatility. It is the method of choice for studying chemical events in heterogeneous systems, which is, after all, what cell-biology is all about.

L+LS: What are some of your career highlights?

PK: I went to Oxford in 1975, and in 1976-77 a group of four of us carried out the first proton NMR experiments to successfully follow metabolism, non-invasively, in whole cells (red blood cells) - that was really magic.

The air was abuzz in 1975 with experiments using phosphorous-31 NMR spectroscopy on intact rat muscle. Everyone believed it would be impossible to use proton NMR to record signals from metabolites in cells because of the utter dominance of the water signal in spectra from biological samples. But the so-called, spin-echo pulse sequence fortuitously enabled us to do this.

Basically, we excited the nuclei of the water molecules with a radiofrequency pulse and then waited a while - as chance would have it, the relaxation time for the water signal was short relative to that of other small mobile molecules, and so by waiting a while we could get the signal from the small molecules. It was remarkable that it worked.

The first NMR spectrometer with a superconducting magnet to be installed in a university was the Bruker 270 megahertz instrument at Oxford, and that was the one we used.

We followed the conversion of glucose to lactic acid in suspensions of human red blood cells - all without smashing the cells up.

Prior to doing those experiments I had met Sir Hans Krebs, the discoverer of the urea cycle and the Krebs cycle.

I had discussed our urea cycle computer model and amongst the things he said about it (apart from declaring he knew little about computers!) was something like: “Ahh, but you don’t know what the enzymes are behaving like inside the cell; it won’t be like the inside of a test-tube!”

We were showing that NMR spectroscopy could be used to measure enzymatic reactions inside cells, and then Sir Hans took great interest in our work. This was another buzz for a boy from the colonies!

The use of the spin-echo pulse sequence for studying metabolism in whole cells was a really important development. It turned out to be a career-forming experiment for me.

L+LS: Have you made a significant biomedical discovery in your research?

PK: There are a few examples of this from my group - being the first to report phospholipase D in human red blood cells provides a good example.

It happened like this...

When I left Oxford at the end of 1978 I took a job at the new medical school at Newcastle University where I set out to combine clinical work, teaching and research, as well as trying to maintain cutting-edge research - I’ve never worked so hard in my life!

Professor Geoff Kellerman, my ‘boss’ at the time, generously organised with Dr Alan Jones, the director of the new National NMR centre at ANU, to allocate me some time on the new 270 megahertz NMR spectrometer. I flew down to Canberra about once a month and Alan and I worked like mad all weekend. An organic chemist by profession, he rapidly became fascinated by the fact that we could study metabolic processes in cells with this instrument. This was an instrument that was really the preserve of organic chemists!

On the strength of reading a New England Journal of Medicine article, which described choline levels being much higher than normal in the red blood cells of patients with bipolar disorder, we began investigating these cells.

Through a clinical biochemist in Newcastle I obtained some blood from patients who were taking lithium carbonate as their main treatment for bipolar disorder. And sure enough the first proton spin-echo NMR spectra showed big choline peaks. In a very facile way we could measure choline concentration in whole red blood cells. We rapidly published a paper on this finding.

But what was the explanation for the high levels of choline? This demanded an explanation!

Radioactive choline transport into red blood cells had been measured by others; and a choline transporter was known to exist, but no-one knew why. It suddenly made sense that this natural process occurred as part of the membrane-turnover in red blood cells and the main reason for the choline transporter was to let the choline out of the cells. But lithium inhibits the choline transporter and, in turn, the choline efflux from the red cells causing it to accumulate.

Thus we discovered that phospholipase D catalyses the removal of the choline head-group from lecithin if lecithin flips to the inside face of the plasma membrane - lecithin normally resides on the outside face of cells. So the choline released inside the cells exits via the choline transporter. All the disparate facts now made sense!

Others have since independently (re)discovered phospholipase D. But our studies led to the hypothesis that lithium may be working in the brain by causing an accumulation of choline that in turn caused elevation of acetylcholine, a neurotransmitter - but the story is not this simple and the mode of action of lithium in bipolar disorder is still not properly understood.

L+LS: Is there an unanswered research question from your career?

PK: Yes, there are many, but a Holy Grail for me is: we don’t know where half of the ATP turnover that occurs in red blood cells goes! And if that’s true for red blood cells, what is it like for other cells in the body, for example, a neuron?

We think we know what the energy requirements are for pumping sodium and potassium ions to maintain membrane potential in a nerve cell, but we must be paying for the shape of the dendrites, dendrite turnover, the trafficking of vesicles within the nerve cell etc, etc.

The balance sheet for energy turnover in cells is not understood.

L+LS: Did your research influence what you taught students at university?

PK: Most definitely!

When I arrived at the University of Sydney in 1980, Dr Greg Ralston and I set about implementing research-led education at least into our third-year classes.

Greg was also interested in new ways of teaching; and we concocted the idea of having ‘Option’ courses where about one-third of the third-year teaching would involve small-group lectures and work groups on topics relevant to each individual academic staff member’s research program. I delivered an Option course on ‘NMR in Biochemistry’ and taught students for several years about applications of NMR, exploring ways of studying reactions in living cells, how fast molecules were exchanging across cell membranes, etc.

We were attracting some of the very best chemistry students to careers in biochemistry, and in the 1980s and 1990s, some very gifted students came my way via chemistry.

Part of the attraction for students was the novelty of NMR in biological applications - it was dynamic and hence very exciting. The technique had reached a stage in its development where one could obtain really fundamental information with relatively simple experiments on cells.

That nexus between chemistry and biochemistry has been broken to a large extent, with the demise of ‘strong’ prerequisites in our undergraduate degrees.

L+LS: What do you think of the current environment for academic staff in the tertiary education system?

PK: There are many more research institutes and specialist research centres than in 1980 when I began my research in Sydney. To maintain a workable balance between teaching, research and organisation is the perennial challenge of an academic. I think academic staff are being put under incredible pressure today. The administration in our universities appears to be becoming top-heavy and very expensive.

I think the recruiting policy for academic staff is flawed. It is demoralising for extant staff who have, in general, worked hard in a department to learn that someone has been brought in from outside (drawing two or three times the average salary) with little or no consultation with the staff. These decisions are often made higher up for reasons of improving research or, less often, educational ‘metrics’.

We must continue emphasising problem-solving as a central aspect of university education. This is being done in many practical classes, but it should be ‘research led’ with specialist courses given by research experts.

L+LS: Can you talk about your time as director of the Singapore Bioimaging Consortium?

PK: This was an unexpected adventure that presented itself at a late stage in my academic career.

In 2009, I was asked to apply for the position as executive director of the Singapore Bioimaging Consortium (SBIC). This was a consortium of scientists dedicated to recording images of animal models of human diseases and how these might change under the influence of various drugs and treatments. I was appointed as incoming executive director in 2010.

I was placed on leave without pay from the university because I had a PhD student still working there - PhD student number 26 as it turns out.

The consortium had been established by Professor Sir George Radda, who had indicated his intention to step down as director at the end of 2010. But he ended up staying on in the SBIC.

The move to such a position in a country like Singapore would be a big change for anybody. The bureaucracy in Singapore is ‘top-down’ and much less democratic than here. I also thought the issue of career development for postdocs needed attention. I tried to implement discussion groups and create a more congenial working environment, but this was difficult to achieve because of a lack of positive feedback on changes that a few senior colleagues and I tried to make.

I had been awarded a 5-year contract but I left after two years and returned to the University of Sydney in 2012. I did not see a way clear to making substantial changes in the research culture of the SBIC and being able to direct my own research projects in the remaining years of my contract to justify fighting on. There were still so many projects and so much unfinished business on my research ‘to-do list’ that returning home was the obvious choice.

Having said that, I’m glad that I went there because it was an incredible watershed for me and led me into dynamic nuclear polarisation that we don’t have time to talk about now!

So, I was appointed Emeritus Professor by the Senate of the University. I secured an ARC Project Grant, which enabled the appointment of a terrific postdoc, and on the strength of that I have a lab and my old office back. So it worked out remarkably well.