Experimental design defended

N 134

TIBS - June 1970

pH 7, it is questionable whether the concentrations of enol-pyruvate in L’I‘c’o are sufficient to cause inhibition. These objections are symptomatic of the difficulties of extrapolating in r,itro enzyme kinetics data to explain situation in ~,ivo: such difficulties have been encountered with other enzyme systems, for example the G6PDH variants 171. However, Kaplan has answered these objections by demonstrating that, in chino, chicken heart LDH (but not chicken muscle LDH) exists in a partially inhibited state [ 11. Thirdly, any theory for the biological value of the LDH isozymes should explain the significance of all five isozymcs and not just the homotetramers. But since the H and M genes may have arisen from gene duplication, it may be argued that the existence of the hybrid forms is an insignificant consequence of the close evolutionary and structural relationships between the H and M gcnc products. Finally, an apparently complete absence of the H type subunit in man does not cause any serious physiological consequences [8]: this is hard to reconcile with the proposed regulatory function of the H4 isozyme. Thus, although the aerobic-anaerobic theory has contributed significantly to understanding the biological role of LDH isozymes, sufficient doubts remain to make it inadvisable to accept this theory as dogma. To add further to the confusion. in

vertebrates, a sixth structurally distinct isozyme, LDH-X, occurs specifically in mature testes and spermatozoa [9, LO]. The ability of this isozyme to utilize a wider range of substrates than the five major isozymes has been suggested to confer the spermatozoa with the metabolic flexibility needed to cope with the wide range of environments encountered in passage from testis to ovum.

Szent-Gyiirgyi’s dogma - hypothesize with a smile SIR: My contribution to the series Textbook Errors concerning the mode of action of lactate dehydrogenase isozymes (7’1BS 4, February, N32) has stimulated replies from Drs Heffron, Huijing and Svasti dealing with different aspects of my article. It is difficult for me to make a brief reply discussing all these aspects. Moreover, it was not my aim to open a debate about the function of lactate dehydrogenase isozymes, but only to point out that the explanations concerning this topic provided by two current textbooks (incidentally I must say two excellent textbooks) are incorrect. These explanations, as well as those provided by most biochemistry textbooks, are based on Kaplan’s hypothesis [l-3] and so, I restricted my discussion to it. Nevertheless I do not mean at all that other points of view do not deserve further consideration, although a general biochemistry textbook will not probably be the appropriate place. I believe that

Kaplan’s hypothesis is still good enough to be taught because it provides a whole and suggestive picture of the question. Of course, it should be emphasized that it cannot be regarded as a dogma. SzentGyiirgyi, one of the pioneers of biochemistry, wrote that the best-founded hypothesis should be posed with a smile. Maybe students should be reminded more often of this statement that I’m sure it can be considered an incontrovertible dogma.

References I Everse. J. and Kaplan, N. 0. (1975) in Isozymcs (Markcrt, C. L.. ed.), Vol. 7. pp. 29-43, Academic Press, New York 2 Cahn. R. D., Kaplan, N. 0.. Levine, L. and Zwilling, E. (19’62) Scrence 136, 962-969 3 Eversc. J. and Kaplan, N. 0. (1973) A& Enzymol. 31. 61-133 4 Hultin. H. 0. (197.5) in Isozymes (Markert. C. L., cd.), Vol. 2, pp. 69-85. Academic Press. New York 5 Fritz. P. J.. White, E. L., Pruitt, K. M. and Vessel. E. S. (I 973) Biochemistry 12, 4034-4039 6 Vessel, E. S. (I 975) in Isozymes (Markert. C. L., cd.). Vol. 2, pp. l-28. Academic Press, NC& York 7 Yoshida, A. (1973) Science 179, 532-537 8 Kitamura. M. and Nishina, J. (1975) in Isozymes (Markert, C. L., ed.). Vol. 2. pp. 97-l 11. Academic Press, New York 9 Blanco. A. and Zinkham, W. H. (1963) Science 139, 60 l-602 10 Goldberg. E. (1963) Scierlcr 139, 602-603 JISNUSON

SVASTI

Department of Biochemistry, Faculty of Science, Mahidol University. Rama VI Road, Bangkok, Thailand.

References I Pesce, A., McKay, R. H., Stolzenbach, F., Cahn. R. D. and Kaplan, N. 0. (1964) J. Bid. Chrm. 239. 1753-1761 2 Dawson, D. M.. Goodfriend. T. L. and Kaplan. N. 0. (1964) Science 143, 929-933 3 Everse. J. and Kaplan, N. 0. (1073) Adv. Enzymol. 37, 61-133 RICARDO

FLORES

Department of Biochemistry, Faculty of Sciences, University of Valencia, Spain. This correspondence

is now closed.

Ion and water transport Experimental

design defended

In TfBS (3. Sept., N210) a Letter to the Editor from Martin J. Kushmerick was published under the title ‘Ion and water transport - experimental design at fault.’ The letter criticizes the paper that M. M. Ochsenfeld and I published five years ago. In this paper we contradicted the earlier publication of Kushmcrick and Podolsky, and we also presented evidence that potassium mobility in normal cytoplasm is reduced by a factor of l/8 when compared to diffusion in an isotonic salt solution. If there is any ‘fault’ it would seem it must have come from Dr Kushmerick’s not having read the literature carefully. The main criticism he offers concerns the effect of a suggested electrical potential gradient. Kushmerick now suggests that we were at fault in ignoring the possible effect of an electrical potential gradient between the cytoplasm and the Ringer solution bathing the cut end. In fact this question was considered and very carcfully dealt with in no less than three different publications. In the kcry article Kushmerick criticized, we had pointed out that no Donnan potential in the longitudinal direction could have existed, because the movement of potassium is across the cut end of the fiber, and the cut end is exposed to a salt concentration roughly equal to that within the cell. However, to make absolutely certain, we dealt with this problem once more. SIR:

(1) If the electrical potential postulated exists, and if it does restrain the movement of cation and artificially reduces its mobility, this effect should be felt by potassium as well as other ions, like sodium. The result would be that the mobility measured from inward diffusion into the cell and outward diffusion from the cell would have totally different values. Using the same EMOC preparation, however, I have shown that the sodium ion mobility is identical within a 20% error, whether mobility of sodium is measured in its inward diffusion into the muscle cytoplasm or in its outward diffusion from the cytoplasm (J. Physiol. (1978) 280, 105-123). (2) Again using the same EMOC preparation I showed that if the electrical potential postulated has an effect in restraining the outward movement of potassium, it should retard the inward movement of anions, like sulfate. I

N 135

demonstrated that the same sulfate diffusion rate. whether these cells retain a normal resting potential in their intact parts or after this resting potential - the seat of the electrical potential Kushmerick refers to - has been obliterated bl. exposure to 0.1 M KCI. Finally in answer to the comment that the potassium measurement Ling and Ochsenfcld reported may include a component due to diffusion in the extracellular space, we would like to point out that if such an error did indeed exist it would make the measured potassium mobility still lower. and this is a further departure from the value Kushmerick and Podolsky reported. In fact, the EMOC preparation was quite effcctivc in pre\.cnting leakage. An exact and thorough investigation of this leakage factor has also been published in the article referred to under (1) above. In summary, we welcome Dr Kushmerick’s question about an important subject, but in view of the abundant evidence to the contrary his concltision that our methods were at fault is regrettable. (;IL.BERT

?%. LlN(i

Director. Dept. of Molecular Biology. Pennsylvania Hospital, Eighth and Spruce Streets. Philadelphia PA 19107, U.S.A.

Cellular energy metabolism Near-equilibrium

model is insufficient

Recently, (TZBS 3, October, 219) EreciAska and Wilson reviewed the evidence that the cytoplasmic [ATPl:[ADP][P,] ratio regulated mitochondrial respiratory rate. This is a valuable concept, but it would also be useful to mention briefly that mitochondrial oxidative capacity is sometimes further controlled by other factors. Ftir example, glucagon may stimulate the activity of the respiratory enzymes independently of exogenous ATP concentrations [I]. Furthermore, GDP regulates respiratory rates of brown adipose tissue mitochondria by modulating membrane conductance [2]. Thyroid hormones also exhibit short-term control of cellular respiration through an interaction with mitochondria which is independent of exogenous adenine nucleotides [3] and protein synthesis [4]. Indeed. thyroxine may elevate the protonic electrochemical potential difference to the point at which there occurs an increase in the ionic conductance of the respiratory membrane [S]. Respiratory stimulation as a consequence of such permeability changes 16.71 is itself a deviation from the model of Erecifiska and Wilson. Finally. certain aspects of brain metabolism ma)

SIR:

Three major cancer prizes presented Henry S. Kaplan, George Klein and Sir Richard Doll have each been presented with one of the three prizes awarded by the General Motors Cancer Research Fund. The prizes, presented in Washington D.C’. on 3 May. each consist of a gold medal and $100,000. Henry Kaplan of Stanford University School of Medicine, U.S.A., has been selected to receive the first Charles F. Kettering Prirc for his research into Hodgkin’s disease, a cancer of the lymph glands. Sir Richard Doll of Oxford University, U.K., was presented with the Charles S. Mott Prize for his work on the cn\ironmental causes of cancer. The third prize, named after Alfred P. Sloan. Jr., has been awarded to George Klein of the Karolinska Institute, Sweden. for his studies on the interrelation of cancer and the immune system in mammal\.

FEBS Prize goes to Carlos Gancedo The winner of this year’s FEBS Anniversary Prize of the Gesellschaft fiir Biologische Chemie is Carlos Gancedo of Madrid. This prize, awarded each year since 1974, is presented to a person under 40 who has been an invited lecturer at a FEBS symposium. Dr Gancedo received the 1979 prize during the closing session of the FEBS enzyme meeting in Dubrovnik, on 21 April. Dr Gancedo, who works at the Instituto de Enzimologia y Patologia Molecular in the Universidad Autcinoma, Madrid, has made important contributions to the study of metabolic regulation, including regulatory interconversion of bacterial glutamine synthetases; glucose-induced ‘catabolite inactivation’ of fructose biphosphatase and phosphoenolpyruvate carboxykinase in yeast; activation of yeast phosphofructokinasc by PI; and the development of methods for in .situ assays of yeast enzymes as a tool for regulation studies.

not be purely controlled by the cytoplasmic [ATP]:[ADP][P,] ratio. During sleep this ratio does not decrease [8] and may even rise [9,10]. However, brain metabolic rate is not inhibited [9], and may even increase during certain sleep periods [11,12]. The data described above are anomalous when considered purely in terms of the model suggested by Ereciliska and Wilson. It is not intended to deprecate such a concept and its general applicability. However, the anomaly is removed if it is accepted that certain physiological regulatory mechanisms arc superimposed upon the control of cellular catabolism bq the cytoplasmic [ATP]:[ADP]IP,] ratio. References 1 Hatestrap, A. P. (197X) R/ocht~m. .I. 172, 399-405 2 Nicholls, D. G. (1976) FERS Lcvr. 61. 103-l IO 3 Iloch. F. L. (1968) Arch. Biorkc~m. Riophw 124, 238-247 4 Bronk, J. R. (1966) Science 153. 63%63Y i Shears. S. B. and Bronk, J. R. (I 97U) Biocl~c~m J. 178, 505-507 6 Nicholls. D. G. (1 Y74) Eur. J. Rioclrrrn. 50. 305-3 15 7 Nicholls, D. G. (1977) E/,r. J. fliocl~rw~. 77, 349-356 t! Reich. P.. Geyer. S. J. and Karnovsky. M. L. (1972) J. Nrurochrm. IV. 4X7-4Y7 Y van den Noort, S. and Brim, K. (I 970) rlrn. J. f’hysiol. 2 1X. 1434-1430 10 Durie. D. J. B., Adam, K., Oswald. I. and Flynn, I. (1978) IRCS Medical Scicwc~e 6. 35 1 11 Brcbbia, D. R. and Attshuler, K. Z. (I 965) Science 150. 1621-1623 I2 Stothers. J. K. and Warner R. M. (lY7X) ./. Phy.,iol. 27X. 435-440 STEPHEN B. SHEARS

Department of Biochemistry, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 YAG. U.K.

Aaron Klug receives Heineken Prize In April the 1979 Heineken Prize was presented to Aaron Klug of the MRC Laboratory of Molecular Biology in Cambridge. U.K. Dr Klug received the Dtl. 100,000 prize in Amsterdam for the pioneering work he and his collaborators have carried out in the study of biological macromolecules, including work on the structure and assembly of viruses, tRNA, chromatin and microtubules. He has also developed methods for reconstructing three-dimensional images from electron micrographs. The Heineken Prize has been awarded every three years since 1964 for exceptional discoveries in the fields of biochemistry and biophysics.