Concepts confused
1
270 TIBS - July 1985 Concepts I do not wish to prolong the discussion of the paper by Bosc~i and Corredor (TIBS 9, 372, 1984) since I basically agree with Porteous (TIBS 10, 14, 1985) on the complexity and multiplicity of control mechanisms. In an article entitled 'Limiting factors in glycolysis of ascites tumor cells', we emphasized many years ago the multiplicity of rate limiting factors (Wu and Racker, J. Biol. Chem. 234, p. 1029, 1959). What I fail to understand is why Dr Porteous 'cannot agree' with me con- cerning the generation of ADP and P~ since I am not talking about flux control but about stoichiometry. We are attempting to identify the ATPases that generate ADP and P~ required for lac- tate production in tumour cells. We are dealing in this case also with a multi- plicity of contributing reactions includ- ing ion transport, biosynthesis, futile cycles, etc. In Ehrlich ascites tumor as well as in several lymphoma cells, ouabain inhibits glycolysis by about 50%. We suggest that under the condi- tions of our assay, the Na+K+-transport- ing ATPase generates a significant portion of the ADP and Pi required for glycolysis. We have recently observed that in rat fibroblasts transfected with the ras oncogene, glycolysis is increased confused 4- to 6-fold compared to the control rat1 cell, is insensitive to ouabain, but is inhibited 50-70% by 10 mM methionine (Racker et al. Proc. Natl Acad. Sci. USA, in press; Boerner et al. Proc. Natl Acad. Sci. USA 82, p. 1350, 1985). In a cell-free homogenate, methionine has no effect on glycolysis. I mention these observations because I do not support Dr Porteous's plea to editors of journals to challenge qualitative observations of this type. I would be delighted if Dr Porteous would apply to these phe- nomena kinetic analyses because I greatly appreciate their importance. On the other hand, I cannot resist pointing out once again the danger of applying clean thinking to dirty systems, par- ticularly while key members of the glycolytic pathway, the contributors to the Pi and ADP pool, remain unknown. EFRAIM RACKER Biochemistry Section, Cornell University, Ithaca, NY, USA. Some useful 'tricks' We read with great interest the article by Dr Gershoni on protein blotting in the March issue of TIBS 1. With respect to the section on directions for the future we would like to point out that recent experiments in our department have shown that: (1) It is possible to renature at least one enzyme, 2',3'-cyclic nucleo- tide 3'-phosphodiesterase, after electro- phoretic transfer from SDS-PAGE gels to nitrocellulose 2. (2) Immunoblotting after protein cleavage can be a powerful method of defining monoclonal anti- body binding sites (epitope analysis) 3. (3) Immunoblotting pre-stalned 2D-PAGE gels is a considerable aid to defining an antigenic protein within a large mixture of proteins4. We suggest that all these are useful 'tricks' to be added to the future reper- toire of protein blotting techniques. References 1 Gershoni, J. M. (1985) Trends Biochem. Sci. 10, 103-11)6 2 Bradbury, J. M. and Thompson, R. J. (1984) B/ochem. J. 221, 361-368 3 Stanley, K. K., Kocher, H-P., Luzio, J. P., Jackson, P. and Tschopp, J. (1985) EMBO J. 4, 375-382 4 Jackson, P. and Thompson, R. J. (1984) Elec- trophoresia 5, 35--42 PETER JACKSON, JANE M. BRADBURY~ and J. PAUL Lt3ZIO Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge CB2 2QR, UK.
Transcript of Concepts confused
270 TIBS - July 1985
Concepts I do not wish to prolong the discussion of the paper by Bosc~i and Corredor (TIBS 9, 372, 1984) since I basically agree with Porteous (TIBS 10, 14, 1985) on the complexity and multiplicity of control mechanisms. In an article entitled 'Limiting factors in glycolysis of ascites tumor cells', we emphasized many years ago the multiplicity of rate limiting factors (Wu and Racker, J. Biol. Chem. 234, p. 1029, 1959).
What I fail to understand is why Dr Porteous 'cannot agree' with me con- cerning the generation of ADP and P~ since I am not talking about flux control but about stoichiometry. We are attempting to identify the ATPases that generate ADP and P~ required for lac- tate production in tumour cells. We are dealing in this case also with a multi- plicity of contributing reactions includ- ing ion transport, biosynthesis, futile cycles, etc. In Ehrlich ascites tumor as well as in several lymphoma cells, ouabain inhibits glycolysis by about 50%. We suggest that under the condi- tions of our assay, the Na+K+-transport - ing ATPase generates a significant portio n of the ADP and Pi required for glycolysis. We have recently observed that in rat fibroblasts transfected with the ras oncogene, glycolysis is increased
c o n f u s e d 4- to 6-fold compared to the control rat1 cell, is insensitive to ouabain, but is inhibited 50-70% by 10 mM methionine (Racker et al. Proc. Natl Acad. Sci. USA, in press; Boerner et al. Proc. Natl Acad. Sci. USA 82, p. 1350, 1985). In a cell-free homogenate, methionine has no effect on glycolysis. I mention these observations because I do not support Dr Porteous's plea to editors of journals to challenge qualitative observations of this type. I would be delighted if Dr Porteous would apply to these phe-
nomena kinetic analyses because I greatly appreciate their importance. On the other hand, I cannot resist pointing out once again the danger of applying clean thinking to dirty systems, par- ticularly while key members of the glycolytic pathway, the contributors to the Pi and ADP pool, remain unknown.
EFRAIM RACKER
Biochemistry Section, Cornell University, Ithaca, NY, USA.
Some useful 'tricks' We read with great interest the article by Dr Gershoni on protein blotting in the March issue of TIBS 1. With respect to the section on directions for the future we would like to point out that recent experiments in our department have shown that: (1) It is possible to renature at least one enzyme, 2',3'-cyclic nucleo- tide 3'-phosphodiesterase, after electro- phoretic transfer from SDS-PAGE gels to nitrocellulose 2. (2) Immunoblotting after protein cleavage can be a powerful method of defining monoclonal anti- body binding sites (epitope analysis) 3. (3) Immunoblotting pre-stalned 2D-PAGE gels is a considerable aid to defining an antigenic protein within a
large mixture of proteins 4. We suggest that all these are useful
'tricks' to be added to the future reper- toire of protein blotting techniques.
References 1 Gershoni, J. M. (1985) Trends Biochem. Sci.
10, 103-11)6 2 Bradbury, J. M. and Thompson, R. J. (1984)
B/ochem. J. 221, 361-368 3 Stanley, K. K., Kocher, H-P., Luzio, J. P.,
Jackson, P. and Tschopp, J. (1985) EMBO J. 4, 375-382
4 Jackson, P. and Thompson, R. J. (1984) Elec- trophoresia 5, 35--42 PETER JACKSON, JANE M. BRADBURY~
and J. PAUL Lt3ZIO Department of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge CB2 2QR, UK.
