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The foibles of congress
8
T 1 B S - J a n u a r y 1 983
Letters to the Editor Glycogen phosphorylase as an ambiquitous enzyme SIR: Further to the article by R. J. Gillies on 'The binding site for aldolase and G3PDH in erythrocyte membranes '~ 1 would like to add a note on the topic of 'ambiquitous' enzymes. Though not specifically mentioned by J, E. Wilson as an 'ambiquitous' enzyme 2, it is well-documented that glycogen phosphorylase is present in sarcoplasmic reticulum microsomal preparations (SR) unless it is removed by a very special treatment. This treatment involves a long-term incubation in a buffer of high ionic strength (see, for example, Ref. 3). However, the relevance of this tight association to the function of either SR or glycogen phosphorylase is not well understood (see, for example, Ref. 4). I wish to emphasize here two striking points of this enzymatic association: First, it does not appear to be rapidly reversible, as is evident from the long-term treatments needed to remove glycogen phosphorylase from SR microsomes. Nevertheless, one should realize that no detailed kinetic study of the dissociation of this enzyme from SR as a function of variables with a priori biological significance, such as AMP, ATP, Ca 2, etc. has, to the best of my knowledge, been published. One should also realize that this pool of glycogen phosphorylase bound to SR probably amounts to 30-40% of the total glycogen phosphorylase of the skeletal muscle celP -8. Surprisingly, this point is given no specific mention in the most widespread reviews of glycogen phosphorylase9-~°. Second, specific glycogen phosphorylase activities of the pool bound to SR are quite low. T. Wiedmer and I have confirmed the presence of a significant amount of relatively inactive glycogen phosphorylase in SR. Total glycogen phosphorylase activity was determined in the presence of 1 mM AMP (as in Ref. 6). Maximum steady-state values ranging from 0.3 to 0.9 ~mols P~(mg glycogen phosphorylase) -~ min -~ at 28°C were obtained with different SR preparations. These values are only slightly smaller than those reported by Entman et al. 4, i.e. 1.5-2.0 p~mols P~(mg glycogen phosphorylase) -t min -~ at 30°C. These activities, however, are far below that of soluble glycogen phosphorylase, a or b forms, under these conditions~'11. To elucidate whether this decreased activity is due to steric hindrance or substrate inaccessibility or alternatively, to
changes in the conformation of this enzyme, I recorded the fluorescence spectrum of the coenzyme pyridoxal-5'phosphate (PLP) in membrane-bound glycogen phosphorylase (C, Gutierrez Merino, unpublished results). The emission band of the PLP spectra centered at approx. 550 nm is practially absent in glycogen phosphorylase bound to SR when excited at 335 nm and only slightly appreciable when excited at 415 nm. PLP fluorescence centered at approx. 550 nm (excitation maxima: 335 and 415 nm) is a typical characteristic of active soluble glycogen phosphorylase due to the fact that PLP is deeply buried in a hydrophobic pocket 12'13. Therefore, it seems that glycogen phosphorylase bound to SR membranes is shifted to a less active conformational state in which PLP is in an environment different to that of the predominant state of soluble native glycogen phosphorylase. In conclusion, the analysis developed here indicates that glycogen phosphorylase shares with the so-called 'ambiquitous' enzymes the major features that characterize this group of enzymes: (i) it can be isolated in either soluble or membrane-bound forms; (ii) the association of the enzyme with SR membranes might be reversible and (iii) the activity and conformationz 1 state of the bound enzyme differs markedly from that of the soluble form. The importance of the function carried out by this
enzyme gives special relevance to this discussion from a regulatory viewpoint.
References 1 Gillies, R. J. (19821 Trends Biochem. Sci. 7, 41-42 2 Wilson, J. E. 119781 Trend~ Biochem. Sci. 3, 124-125 3 Meissner~ G. 119751 Biochim. Biophys. Acta
389, 51-68 4 Entman, M. L., Keslensky, S. S., Chu, A. and Van Winkle, W. B. (19801J. Biol. ('hem. 255, 6245-6252 5 MacLennan, D. H. 1197111J.Biol. (_'hem. 245, 4508-4518 6 Gutierrez-Merino, C., GarciaBlanco,F., Pocovi, M., Menendez, M. and Laynez, J. 11980) J. Biochem. 87, 148.3-1491/ 7 Michalak,M., Campbell, K. P. and MacLennan. D. H. (1980)J. Biol. Chem. 255, 1317-1326 8 Stewart, P. S. and MacLennan, D. H. (1974) J. Biol. Chem. 249, 985-993 9 Graves, D. J. and Wang, J. H. 119721 in The Enzymes (Boyer, P. D., ed.), 3rd edn, Vol. Vll, pp. 436482, AcademicPress 10 Busby,S. J. W. andRadda,G K. (1976)inCurrent Topics of Cellular Regulation (Horecker, B.L. and Stadtman, E.R., eds), Vol. 10, pp. 89-160, AcademicPress ! l Kasvinsky,P. J., Madsen, N. B., Sygush, J. and Fletterick, R.J. 11978)J. Biol. (_'hem. 253, 3343--3351 12 Cortijo, M., Steinberg, I.Z. and Shaltiel, S. (19711J. Biol. (_'hem. 246, 933-938 13 Shaltiel, S. and Cortijo, M. (19701 Biochim. Biophys. Res. Comm. 4 I, 594---6011 CARLOSGUTIERREZMERINO Facultad de Medicina, Dpto. de Fisiologia, Universidad de Extremadura, Badajoz, Spain.
The foibles of Congress SIR: I admit not understanding the workings of Congress. Does anybody ? The Congressional Fellow, Dr CookDeegan [T1BS 119821 Vol. 7, Dec., p. 434], is overawed but does not know the real way our elected representatives foist their demands on federal staff. I cited the case of Senator Douglas pressuring that great public servant, Dr Endicott. I could cite dozens more from my personal experience. As to setting national goals for science, you have to be acquainted with the history of the development of science, If the question of building the atom bomb had been placed before the Congress when they were preoccupied with the superbomb of carbon-oxygen, the outcome would have been disastrous. To cite the assessment of a practical man, Admiral Leahey, Roosevelt's military
adviser: 'Money is being wasted on the fuzzy-headed, soft-brained professors. It will never work.' 1 What kind of national goals was Congress setting prior to World War II when the American people spent more on funeral flowers than the American govemment spent on medical research?
Reference I Mosely,L. 119821Marshall, Hero of Our Times, HearstBooks ERNEST BOREK Chairman, Department of Molecular Biology, AMC Cancer Research Center and Hospital, 6401 West Colfax Avenue, Lakewood, CO 80214, U.S.A.
T 1 B S - J a n u a r y 1 983
Letters to the Editor Glycogen phosphorylase as an ambiquitous enzyme SIR: Further to the article by R. J. Gillies on 'The binding site for aldolase and G3PDH in erythrocyte membranes '~ 1 would like to add a note on the topic of 'ambiquitous' enzymes. Though not specifically mentioned by J, E. Wilson as an 'ambiquitous' enzyme 2, it is well-documented that glycogen phosphorylase is present in sarcoplasmic reticulum microsomal preparations (SR) unless it is removed by a very special treatment. This treatment involves a long-term incubation in a buffer of high ionic strength (see, for example, Ref. 3). However, the relevance of this tight association to the function of either SR or glycogen phosphorylase is not well understood (see, for example, Ref. 4). I wish to emphasize here two striking points of this enzymatic association: First, it does not appear to be rapidly reversible, as is evident from the long-term treatments needed to remove glycogen phosphorylase from SR microsomes. Nevertheless, one should realize that no detailed kinetic study of the dissociation of this enzyme from SR as a function of variables with a priori biological significance, such as AMP, ATP, Ca 2, etc. has, to the best of my knowledge, been published. One should also realize that this pool of glycogen phosphorylase bound to SR probably amounts to 30-40% of the total glycogen phosphorylase of the skeletal muscle celP -8. Surprisingly, this point is given no specific mention in the most widespread reviews of glycogen phosphorylase9-~°. Second, specific glycogen phosphorylase activities of the pool bound to SR are quite low. T. Wiedmer and I have confirmed the presence of a significant amount of relatively inactive glycogen phosphorylase in SR. Total glycogen phosphorylase activity was determined in the presence of 1 mM AMP (as in Ref. 6). Maximum steady-state values ranging from 0.3 to 0.9 ~mols P~(mg glycogen phosphorylase) -~ min -~ at 28°C were obtained with different SR preparations. These values are only slightly smaller than those reported by Entman et al. 4, i.e. 1.5-2.0 p~mols P~(mg glycogen phosphorylase) -t min -~ at 30°C. These activities, however, are far below that of soluble glycogen phosphorylase, a or b forms, under these conditions~'11. To elucidate whether this decreased activity is due to steric hindrance or substrate inaccessibility or alternatively, to
changes in the conformation of this enzyme, I recorded the fluorescence spectrum of the coenzyme pyridoxal-5'phosphate (PLP) in membrane-bound glycogen phosphorylase (C, Gutierrez Merino, unpublished results). The emission band of the PLP spectra centered at approx. 550 nm is practially absent in glycogen phosphorylase bound to SR when excited at 335 nm and only slightly appreciable when excited at 415 nm. PLP fluorescence centered at approx. 550 nm (excitation maxima: 335 and 415 nm) is a typical characteristic of active soluble glycogen phosphorylase due to the fact that PLP is deeply buried in a hydrophobic pocket 12'13. Therefore, it seems that glycogen phosphorylase bound to SR membranes is shifted to a less active conformational state in which PLP is in an environment different to that of the predominant state of soluble native glycogen phosphorylase. In conclusion, the analysis developed here indicates that glycogen phosphorylase shares with the so-called 'ambiquitous' enzymes the major features that characterize this group of enzymes: (i) it can be isolated in either soluble or membrane-bound forms; (ii) the association of the enzyme with SR membranes might be reversible and (iii) the activity and conformationz 1 state of the bound enzyme differs markedly from that of the soluble form. The importance of the function carried out by this
enzyme gives special relevance to this discussion from a regulatory viewpoint.
References 1 Gillies, R. J. (19821 Trends Biochem. Sci. 7, 41-42 2 Wilson, J. E. 119781 Trend~ Biochem. Sci. 3, 124-125 3 Meissner~ G. 119751 Biochim. Biophys. Acta
389, 51-68 4 Entman, M. L., Keslensky, S. S., Chu, A. and Van Winkle, W. B. (19801J. Biol. ('hem. 255, 6245-6252 5 MacLennan, D. H. 1197111J.Biol. (_'hem. 245, 4508-4518 6 Gutierrez-Merino, C., GarciaBlanco,F., Pocovi, M., Menendez, M. and Laynez, J. 11980) J. Biochem. 87, 148.3-1491/ 7 Michalak,M., Campbell, K. P. and MacLennan. D. H. (1980)J. Biol. Chem. 255, 1317-1326 8 Stewart, P. S. and MacLennan, D. H. (1974) J. Biol. Chem. 249, 985-993 9 Graves, D. J. and Wang, J. H. 119721 in The Enzymes (Boyer, P. D., ed.), 3rd edn, Vol. Vll, pp. 436482, AcademicPress 10 Busby,S. J. W. andRadda,G K. (1976)inCurrent Topics of Cellular Regulation (Horecker, B.L. and Stadtman, E.R., eds), Vol. 10, pp. 89-160, AcademicPress ! l Kasvinsky,P. J., Madsen, N. B., Sygush, J. and Fletterick, R.J. 11978)J. Biol. (_'hem. 253, 3343--3351 12 Cortijo, M., Steinberg, I.Z. and Shaltiel, S. (19711J. Biol. (_'hem. 246, 933-938 13 Shaltiel, S. and Cortijo, M. (19701 Biochim. Biophys. Res. Comm. 4 I, 594---6011 CARLOSGUTIERREZMERINO Facultad de Medicina, Dpto. de Fisiologia, Universidad de Extremadura, Badajoz, Spain.
The foibles of Congress SIR: I admit not understanding the workings of Congress. Does anybody ? The Congressional Fellow, Dr CookDeegan [T1BS 119821 Vol. 7, Dec., p. 434], is overawed but does not know the real way our elected representatives foist their demands on federal staff. I cited the case of Senator Douglas pressuring that great public servant, Dr Endicott. I could cite dozens more from my personal experience. As to setting national goals for science, you have to be acquainted with the history of the development of science, If the question of building the atom bomb had been placed before the Congress when they were preoccupied with the superbomb of carbon-oxygen, the outcome would have been disastrous. To cite the assessment of a practical man, Admiral Leahey, Roosevelt's military
adviser: 'Money is being wasted on the fuzzy-headed, soft-brained professors. It will never work.' 1 What kind of national goals was Congress setting prior to World War II when the American people spent more on funeral flowers than the American govemment spent on medical research?
Reference I Mosely,L. 119821Marshall, Hero of Our Times, HearstBooks ERNEST BOREK Chairman, Department of Molecular Biology, AMC Cancer Research Center and Hospital, 6401 West Colfax Avenue, Lakewood, CO 80214, U.S.A.