Photosynthesis Research 46: 45-46, 1995. t~) 1995 Kluwer Academic Publishers. Printed in the Netherlands. Historical corner
One thing leading to another David A. Walker 6, Biddlestone Village, Netherton, Northumberland NE65 7DT, UK
Received5 March 1995; acceptedin revised form 23 March 1995
Key words: phosphoenolpyruvate carboxylase, photophosphorylation, photosynthesis, respiration, sugar biosyn-
thesis
For some time in the 1950s, Arnon, Whatley et al. occupied most of my waking thoughts. In my scientific apprenticeship I had been more than fortunate in my mentors. In Harry Beevers' laboratory at Purdue I had learned how to isolate highly active mitochondria from castor beans. My PhD (on the probable role of phosphoenolpyruvate carboxylase in Crasssulacean plants) was with Meirion Thomas at Newcastle. An invitation to work with Robin Hill at Cambridge had followed. By then, Arnon had ensured that photosynthetic phosphorylation was all the rage. Cyclic was exciting enough but non-cyclic was positively mind boggling. Like others of my generation, I had become accustomed to the idea that the oxidation of reduced 'DPN' was accompanied by the esterification of ADP to yield ATP. Conversely, ATP formation accompanying 'TPN' reduction (Arnon 1961) seemed remarkably like getting water to run up hill. Only the subsequent enunciation of the Z-scheme, by Hill and Bendall permitted the comforting analogy that the water still ran down hill but that it was lifted up, before and after, by two separate photochemical reactions (a matter not always viewed by Dan Arnon with total enthusiasm). However, while epoch-making papers continued to pour out of the Arnon lab, I prepared 'chloroplasts' in Arnon's mandatory Tris-NaC1 medium. These 'chloroplasts' did all that was claimed of them, and more, but confirmation of the results of others is not the peak of a young scientist's ambition. I remember particularly how crestfallen I felt one Saturday evening when, after spending long hours at last establishing what I believed to be a really new fact, I went into the library only to discover that its essence was printed in that day's Nature. Berkeley still reigned supreme. Similarly while we were able to come up with pyocyanine as
a spectacularly effective initiator of cyclic photophosphorylation, Hill never found an opportunity to prepare a new batch of (what he had originally defined as) 'methaemoglobin reducing factor'. Soon it was too late in the sense that Arnon's renowned ferredoxin contributions started to enter the public domain. So these were the usual sorts of scientific disappointments and, although the Cambridge Biochemistry Department was Mecca, my own modest venture into photophosphorylation, which involved endless repetitive pipetting into reaction mixtures, was drudgery of the first order. Escape to a lectureship at Queen Mary College (London) therefore came as something of a relief. There I might have continued to work happily in the border lands between photosynthesis and respiration had Charles Whittingham (then my boss) not insisted that I should try and isolate chloroplasts which would fix CO2. Of course Arnon, Whatley et al. had already done this but, although they correctly anticipated that improved rates would surely follow, those that they reported at the time fell short of what could be observed in vivo. Arnon and his colleagues had prepared 'chloroplasts' in a Tris-NaCl medium because they wished to avoid any possibility that ATP could be formed in their experiments as a consequence of oxidative degradation of sugar. Since photophosphorylation was now well established I was freed of this particular constraint and returned to Hill's well tried osmoticum of third molar sugar. I favoured sorbitol, phosphate (as a buffer) and one or two other additives and procedures which ultimately permitted the separation of intact chloroplasts (i.e., chloroplasts shown to be surrounded by a double envelope) which could perform as well as the parent tissue if supplemented with catalytic amounts of triose phosphates. While working on this latter requirement
46 at Imperial College, with Bill Cockburn and the late Carl Baldry, I was devastated to learn that Dick Jensen and A1 Bassham had just managed to achieve rates as good or better than ours but without any requirement for added Calvin cycle intermediates. Chagrin abounded; what had I missed? In the end I suppose that the answer was worth the chagrin. The key factor turned out to be Jensen's use of inorganic pyrophosphate. Baldry, Cockburn and I were well aware that, whereas orthophosphate had to be present in reaction mixtures in order to achieve CO2 dependent oxygen evolution by intact chloroplasts, it was inhibitory in larger amounts and that this inhibition could be reversed by the addition of triose phosphates or phosphoglycerate. What we did not know until then was that inorganic pyrophosphate protected against orthophosphate inhibition. Moreover because pyrophosphate did not cross the chloroplast envelope and our preparations contained both magnesium and pyrophosphatase (released from ruptured chloroplasts), pyrophosphate also gave rise to orthophosphate in optimally small quantities. But what did all of this mean? It should be remembered that at this time the inability of isolated chloroplasts to make sucrose (a finding which did not go unchallenged) was widely regarded as a defect. Nevertheless it seemed inescapable that our chloroplasts were fully functional and that what we were seeing was a manifestation of transport across envelopes. Accordingly, in a contemporary review (Walker and
Crofts 1970), I wrote 'if sugar phosphates are exported from the chloroplast there must be a corresponding import of phosphate (in some form) if steady state photosynthesis is to be maintained ...... A direct obligatory exchange between orthophosphate (outside) and sugar phosphate (inside) could account for the inhibition of photosynthesis by orthophosphate and its reversal by sugar phosphates'. Happily, this rash proposal was soon to be fully vindicated by Hans Heldt as the principal attribute of what is now known as 'the phosphate translocator'. For those who look for morals there is the inescapable fact that, as Arnon repeatedly insisted at the outset of these linked events, it is important to pay careful attention to the likely consequences of adding materials to reaction mixtures in order to maintain osmotic pressure, pH etc. Ironically, if everyone concerned had been even more careful in what they added it is perhaps unlikely that we would be quite as well informed about these aspects of photosynthesis as we are now.
References
Amon DI (1961) Cell-freephotosynthesisand the energyconversion process. In: McElroy WD and Kass B (eds) Light and Life, pp 489-566. Johns Hopkins Press, Baltimore, MD WalkerDAand CroftsAR (1970)Photosynthesis.Ann Rev Biochem 39:389-428
One thing leading to another David A. Walker 6, Biddlestone Village, Netherton, Northumberland NE65 7DT, UK
Received5 March 1995; acceptedin revised form 23 March 1995
Key words: phosphoenolpyruvate carboxylase, photophosphorylation, photosynthesis, respiration, sugar biosyn-
thesis
For some time in the 1950s, Arnon, Whatley et al. occupied most of my waking thoughts. In my scientific apprenticeship I had been more than fortunate in my mentors. In Harry Beevers' laboratory at Purdue I had learned how to isolate highly active mitochondria from castor beans. My PhD (on the probable role of phosphoenolpyruvate carboxylase in Crasssulacean plants) was with Meirion Thomas at Newcastle. An invitation to work with Robin Hill at Cambridge had followed. By then, Arnon had ensured that photosynthetic phosphorylation was all the rage. Cyclic was exciting enough but non-cyclic was positively mind boggling. Like others of my generation, I had become accustomed to the idea that the oxidation of reduced 'DPN' was accompanied by the esterification of ADP to yield ATP. Conversely, ATP formation accompanying 'TPN' reduction (Arnon 1961) seemed remarkably like getting water to run up hill. Only the subsequent enunciation of the Z-scheme, by Hill and Bendall permitted the comforting analogy that the water still ran down hill but that it was lifted up, before and after, by two separate photochemical reactions (a matter not always viewed by Dan Arnon with total enthusiasm). However, while epoch-making papers continued to pour out of the Arnon lab, I prepared 'chloroplasts' in Arnon's mandatory Tris-NaC1 medium. These 'chloroplasts' did all that was claimed of them, and more, but confirmation of the results of others is not the peak of a young scientist's ambition. I remember particularly how crestfallen I felt one Saturday evening when, after spending long hours at last establishing what I believed to be a really new fact, I went into the library only to discover that its essence was printed in that day's Nature. Berkeley still reigned supreme. Similarly while we were able to come up with pyocyanine as
a spectacularly effective initiator of cyclic photophosphorylation, Hill never found an opportunity to prepare a new batch of (what he had originally defined as) 'methaemoglobin reducing factor'. Soon it was too late in the sense that Arnon's renowned ferredoxin contributions started to enter the public domain. So these were the usual sorts of scientific disappointments and, although the Cambridge Biochemistry Department was Mecca, my own modest venture into photophosphorylation, which involved endless repetitive pipetting into reaction mixtures, was drudgery of the first order. Escape to a lectureship at Queen Mary College (London) therefore came as something of a relief. There I might have continued to work happily in the border lands between photosynthesis and respiration had Charles Whittingham (then my boss) not insisted that I should try and isolate chloroplasts which would fix CO2. Of course Arnon, Whatley et al. had already done this but, although they correctly anticipated that improved rates would surely follow, those that they reported at the time fell short of what could be observed in vivo. Arnon and his colleagues had prepared 'chloroplasts' in a Tris-NaCl medium because they wished to avoid any possibility that ATP could be formed in their experiments as a consequence of oxidative degradation of sugar. Since photophosphorylation was now well established I was freed of this particular constraint and returned to Hill's well tried osmoticum of third molar sugar. I favoured sorbitol, phosphate (as a buffer) and one or two other additives and procedures which ultimately permitted the separation of intact chloroplasts (i.e., chloroplasts shown to be surrounded by a double envelope) which could perform as well as the parent tissue if supplemented with catalytic amounts of triose phosphates. While working on this latter requirement
46 at Imperial College, with Bill Cockburn and the late Carl Baldry, I was devastated to learn that Dick Jensen and A1 Bassham had just managed to achieve rates as good or better than ours but without any requirement for added Calvin cycle intermediates. Chagrin abounded; what had I missed? In the end I suppose that the answer was worth the chagrin. The key factor turned out to be Jensen's use of inorganic pyrophosphate. Baldry, Cockburn and I were well aware that, whereas orthophosphate had to be present in reaction mixtures in order to achieve CO2 dependent oxygen evolution by intact chloroplasts, it was inhibitory in larger amounts and that this inhibition could be reversed by the addition of triose phosphates or phosphoglycerate. What we did not know until then was that inorganic pyrophosphate protected against orthophosphate inhibition. Moreover because pyrophosphate did not cross the chloroplast envelope and our preparations contained both magnesium and pyrophosphatase (released from ruptured chloroplasts), pyrophosphate also gave rise to orthophosphate in optimally small quantities. But what did all of this mean? It should be remembered that at this time the inability of isolated chloroplasts to make sucrose (a finding which did not go unchallenged) was widely regarded as a defect. Nevertheless it seemed inescapable that our chloroplasts were fully functional and that what we were seeing was a manifestation of transport across envelopes. Accordingly, in a contemporary review (Walker and
Crofts 1970), I wrote 'if sugar phosphates are exported from the chloroplast there must be a corresponding import of phosphate (in some form) if steady state photosynthesis is to be maintained ...... A direct obligatory exchange between orthophosphate (outside) and sugar phosphate (inside) could account for the inhibition of photosynthesis by orthophosphate and its reversal by sugar phosphates'. Happily, this rash proposal was soon to be fully vindicated by Hans Heldt as the principal attribute of what is now known as 'the phosphate translocator'. For those who look for morals there is the inescapable fact that, as Arnon repeatedly insisted at the outset of these linked events, it is important to pay careful attention to the likely consequences of adding materials to reaction mixtures in order to maintain osmotic pressure, pH etc. Ironically, if everyone concerned had been even more careful in what they added it is perhaps unlikely that we would be quite as well informed about these aspects of photosynthesis as we are now.
References
Amon DI (1961) Cell-freephotosynthesisand the energyconversion process. In: McElroy WD and Kass B (eds) Light and Life, pp 489-566. Johns Hopkins Press, Baltimore, MD WalkerDAand CroftsAR (1970)Photosynthesis.Ann Rev Biochem 39:389-428
