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Control of Ca2+ pumps
242
"ITBS - J u l y 1982
that was old so-and-so, the - !') surely does more harm than openly knowing so-andso's views and being able to discuss them with him. Anyway, some authors actually appreciate the referee's comments! Perhaps editors should allow referees to choose between revealing or concealing their identities: they might consider this choice in deciding whom to re-select as referees. J. R. BAKER The Institute of Terrestrial Ecology, The Culture Centre of Algae and Protozoa, 36 Storey's Way, Cambridge, U.K.
Control of Ca 2+ pumps sin: The article by R. H. Michell 'Two sites for Ca 2~control in one Ca 2~pump' in T I B S (April, 1982, 123) informs a general audience on the activating effects of limited
proteolysis and lipid modifications on the red cell calcium pump. It also mentions the calmodulin-like action of mild proteolysis which seems to remove a control site from the pump polypeptide, decreasing the molecular weight of the transport protein. However, the two cited studies, by the Z/Jrich and Bern groups, were published in 1981, while Taverna and Hanahan 1and our group2,:~reported such findings in 1980. In our Cell Calcium papers, reporting studies on the calcium pump in inside--out red cell membrane vesicles (IOVs), we stated: 'Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters - that is converts the system to a "high calcium-affinity" state'. And 'In trypsin-digested IOVs the molecular weight of the :~2P-labelled EP is shifted to lower values (110--120,000). We suggest that trypsin digestion cleaves off a
Making electrophoresis buffers sin: The pH meter has revolutionized biochemistry. It's now unnecessary to think when you make buffers. Unfortunately, 'not thinking' has occasionally caused me grief. Electrophoresis buffers have caused the most trouble. I easily forget that salt concentration is more important than pH in making a reproducible buffer. For example when I make tris buffers at slightly differing temperatures I often forget to keep the tris chloride concentration, rather than the pH, constant. I'm reasonably careful with the pH meter, but my buffers may vary by O. 1 pH unit. When I adjust the voltage on my power supply and discover that the current is not the same as it was last time, i'm reminded that 0.1 pH unit variation can be
25% variation in salt concentration. Occasionally the salt concentration of my Laemmli resolving gel buffer has been so low that 0.1% SDS has been below the critical micelle concentration and I've gotten fuzzy protein bands. It's easy to forget that the pH scale is logarithmic and that salt concentration errors are the exponent of pH errors. Fortunately, the problem is easily solved. If you're a chemist with reasonable knowledge of electrodes and solution thermodynamics you can chuckle at my ineptitude with pH meters. If you're like me, simply measure your buffer components by weight or volume. Pipets and balances are accurate, thermostable, insensitive to specific salt effects and reliable. (When did
20-40,000 tool. wt calmodulin-binding regulatory subunit of the calcium pump molecule'. These studies were then reinforced and extended by the ZiJrich and Bern groups in isolated calcium pump protein preparations. Both groups largely referred to our work in this subject. G. GARDOS B. SARKADI AGNESENYEDI Department of Cell Metabolism, National Institute of Haematology and Blood Transfusion, Budapest, Hungary
References I Taverna, R. D. and Hanahan, D. V. (1980) Biochem. Biophys. Res. (~mmun. 94. 652-659 2 Sarkadi, B., Enyedi,A. andGfirdos.G. (1980)('ell Calcium I. 287-297 3 Enyedi, A., Sarkardi, B., Szasz. i.. Bot. G. and G',irdos,G. (1980)('ell (ah'ium 1,299-310
you last calibrate yours?). My tris buffers are more reproducible when I dispense concentrated hydrochloric acid (assuming 11.6 M) with a blowout pipet than when ! adjust to a fixed pH with the meter. When I carefully weigh and dilute buffer components and then find the pH to be 'unusual' it's easy to forget that the pH meter and not the buffer probably needs adjustment. I hope that what I've said is trivial. If so, remember that for every person like you there's probably another who has no idea what I've been talking about. If you're unlucky he's in your lab right now making up buffers. LESLIEC. LANE Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE 68583-0722 U.S.A.
"ITBS - J u l y 1982
that was old so-and-so, the - !') surely does more harm than openly knowing so-andso's views and being able to discuss them with him. Anyway, some authors actually appreciate the referee's comments! Perhaps editors should allow referees to choose between revealing or concealing their identities: they might consider this choice in deciding whom to re-select as referees. J. R. BAKER The Institute of Terrestrial Ecology, The Culture Centre of Algae and Protozoa, 36 Storey's Way, Cambridge, U.K.
Control of Ca 2+ pumps sin: The article by R. H. Michell 'Two sites for Ca 2~control in one Ca 2~pump' in T I B S (April, 1982, 123) informs a general audience on the activating effects of limited
proteolysis and lipid modifications on the red cell calcium pump. It also mentions the calmodulin-like action of mild proteolysis which seems to remove a control site from the pump polypeptide, decreasing the molecular weight of the transport protein. However, the two cited studies, by the Z/Jrich and Bern groups, were published in 1981, while Taverna and Hanahan 1and our group2,:~reported such findings in 1980. In our Cell Calcium papers, reporting studies on the calcium pump in inside--out red cell membrane vesicles (IOVs), we stated: 'Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters - that is converts the system to a "high calcium-affinity" state'. And 'In trypsin-digested IOVs the molecular weight of the :~2P-labelled EP is shifted to lower values (110--120,000). We suggest that trypsin digestion cleaves off a
Making electrophoresis buffers sin: The pH meter has revolutionized biochemistry. It's now unnecessary to think when you make buffers. Unfortunately, 'not thinking' has occasionally caused me grief. Electrophoresis buffers have caused the most trouble. I easily forget that salt concentration is more important than pH in making a reproducible buffer. For example when I make tris buffers at slightly differing temperatures I often forget to keep the tris chloride concentration, rather than the pH, constant. I'm reasonably careful with the pH meter, but my buffers may vary by O. 1 pH unit. When I adjust the voltage on my power supply and discover that the current is not the same as it was last time, i'm reminded that 0.1 pH unit variation can be
25% variation in salt concentration. Occasionally the salt concentration of my Laemmli resolving gel buffer has been so low that 0.1% SDS has been below the critical micelle concentration and I've gotten fuzzy protein bands. It's easy to forget that the pH scale is logarithmic and that salt concentration errors are the exponent of pH errors. Fortunately, the problem is easily solved. If you're a chemist with reasonable knowledge of electrodes and solution thermodynamics you can chuckle at my ineptitude with pH meters. If you're like me, simply measure your buffer components by weight or volume. Pipets and balances are accurate, thermostable, insensitive to specific salt effects and reliable. (When did
20-40,000 tool. wt calmodulin-binding regulatory subunit of the calcium pump molecule'. These studies were then reinforced and extended by the ZiJrich and Bern groups in isolated calcium pump protein preparations. Both groups largely referred to our work in this subject. G. GARDOS B. SARKADI AGNESENYEDI Department of Cell Metabolism, National Institute of Haematology and Blood Transfusion, Budapest, Hungary
References I Taverna, R. D. and Hanahan, D. V. (1980) Biochem. Biophys. Res. (~mmun. 94. 652-659 2 Sarkadi, B., Enyedi,A. andGfirdos.G. (1980)('ell Calcium I. 287-297 3 Enyedi, A., Sarkardi, B., Szasz. i.. Bot. G. and G',irdos,G. (1980)('ell (ah'ium 1,299-310
you last calibrate yours?). My tris buffers are more reproducible when I dispense concentrated hydrochloric acid (assuming 11.6 M) with a blowout pipet than when ! adjust to a fixed pH with the meter. When I carefully weigh and dilute buffer components and then find the pH to be 'unusual' it's easy to forget that the pH meter and not the buffer probably needs adjustment. I hope that what I've said is trivial. If so, remember that for every person like you there's probably another who has no idea what I've been talking about. If you're unlucky he's in your lab right now making up buffers. LESLIEC. LANE Department of Plant Pathology, University of Nebraska-Lincoln, Lincoln, NE 68583-0722 U.S.A.