I5
YEARSAGO something
This is the nineteenth in a series of articles recalling scien4fic events in FASEB 50 years ago, including reminiscences of the 1942 Annual Meeting.
On
the Initiation and Starch William
of Glycogen Synthesis J.
Whelan
It was exactly 50 years ago this month that I entered the University of Birmingham, U.K., as a 17-year-old undergraduate student, majoring in chemistry and supported by a scholarship from the Brewer’s Society, which wanted to bring more science into brewing. To that end, it was my ambition to become a biochemist, but wartime regulations prevented me from majoring in that subject. The plan that was put to me was to pursue chemistry, which was a large component of the biochemistry degree course (then called industrial fermentation), and perhaps by then the war would be over and I could make the switch. However, going through an accelerated curriculum, I graduated before the war ended, and after working for the Royal Navy on antisubmarine devices, I was able to pursue biochemical research in the same Department of Chemistry. The head of the Department was Sir Norman Haworth, the 1937 Nobel Laureate in Chemistry. He and his close associate, Stanley Peat, had gradually moved from strictly chemical to biochemical studies of starch, inspired by the work of Charles Hanes (1). In characterizing potato phosphorylase and carrying out the in vitro synthesis of starchlike material, Hanes in turn had received his inspiration from the work of Carl and Gerty Con and others, who had described mammalian phosphorylase and its ability to synthesize glycogen from glucose 1-phosphate (the Con ester) (2). I became interested in studying the mechanism of action of starch phosphorylase under the guidance of Peat, my thesis mentor. The connection of this account with the science of 1942 is that volume 147 of the Journal of Biological Chemistry contained two papers that were to be very important in my own researches. Arda Green and Con and Con (3) reported in a brief communication the crystallization of muscle phosphonylase and noted that for the crystalline enzyme to utilize glucose 1-phosphate in the synthesis of glycogen-like material,
there
was
the
same
dependency
on
added
glycogen
that they had reported earlier for less pure preparations of the enzyme (2). In other words, the synthesis of glycogen from glucose 1-phosphate depended on the addition of a trace of glycogen. The second paper was a full-length report by David Green and Paul Stumpf (4)-Green, in the unfamiliar role of describing the properties of starch phosphonylase, whereas we think of him for his enormous contributions over the next several decades in the field of bioenergetics. Green and Stumpf described many intriguing properties of potato phosphorylase and their paper became one that I consulted again and again. They showed that glycogen, starch, and dextrins derived therefrom were necessary to “catalyze” the polymerization of glucose from glucose 1-phosphate. They regarded the “catalyst” as literally that,
3218
that
accepted
phosphate
from
the Con
ester
and
then discharged it as inorganic phosphate. Con and Con (2) and Hanes (1) had referred to the phenomenon as “activation?’ The later concept that the role of the added carbohydrate in assisting starch (glycogen) synthesis was that of a primer, receiving glucose units from glucose 1-phosphate and becoming part of the finished macromolecule, had not been grasped, but had been by 1950 when we were able to show that of the oligosacchanide series based on glycogen and starch, maltotriose is the smallest that will accept glucose units in this way in the synthesis catalyzed by potato phosphorylase (5). The story of the search, through 1985, for the endogenous primer for glycogen and starch synthesis has been told elsewhere (6, 7). For glycogen, the details have emerged only in the last few years. For starch, the definitive answer is still not _______ at hand (8). The breakthrough came in the 1970s when Clara Krisman (9) reported her ability, with liver extracts, to synthesize glycogen-like material bound to protein. Following up this clue, we reported in 1985 the isolation of a covalently bound protein from muscle glycogen in one molecular proportion to each glycogen molecule. We named it glycogenin (10). The first alpha-glucose residue of glycogen, on which the synWilliam J. Whelan thesis of the molecule is initi-
________
ated, proved to be linked to glycogenin through a novel carbohydrate-to-protein bond, involving the hydroxyl group of a tyrosine residue (11-13). Next, glycogen-free glycogenin was isolated from a muscle extract and found to be an autocatalytic enzyme, creating a maltosaccharide primer on itself (14, 15). This does not direclty prime muscie-glycogen (macroglycogen, Mr lO7Da) synthesis, but that of a stable intermediate, proglycogen (Mr approx. 400 Da) (16), which functions both in the synthesis and degradation of macroglycogen (17). The more recent advances have been reviewed by Philip Cohen (18), whose laboratory has made important contributions, including the sequencing of glycogenin. Glycogen and starch were the first naturally occurring macromolecules to be synthesized in a cell-free system (1, 2), long before proteins or nucleic acids. But it was to be another 50 years before the nature of the initiation step for glycogen synthesis was understood. We had been correct, in our search for the endogenous primer, in assuming that it would be a maltosacchanide. We had been incorrect in looking for a system to synthesize a free oligosaccharide. Instead, it proved to be an autocatalytically synthesized, protein-bound maltosaccharide (malto-octaose, refs. 13, 19), which at the same time explained the reason for the occurrence of protein in glycogen, first reported 100 years ago (20). The answer to the nature of the primer was there all the time, inside the glycogen molecule, waiting to be uncovered.
Correspondence may be addressed to Dr. Whelan at the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, P.O. Box 016129, Miami, FL 33101, USA.
50 YEARS AGO
Vol. 6
October 1992
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 16, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
REFERENCES 1. Hanes, C. 5. (1940) The reversible formation of starch from glucose-i-phosphate catalyzed by potato phosphorylase Proc. Roy. Soc. Lond. B129, 174-208 2. Con, G. T., and Con, C. F. (1940) The kinetics of the enzymatic synthesis of glycogen from glucose-l-phosphate. j BioL Chem. 135, 733-756 3. Green, A. A., Con, G. T., and Con, C. F (1940) Crystalline muscle phosphonylase. J BioL Chem. 142, 447-448 4. Green, D. E., and Stumpf, P. K. (1942) Starch phosphorylase of potato.]. BioL Chem. 142, 355-366 5. Bailey, J. M., Whelan, W. J., and Peat, S. (1950) Carbohydrate primers in the synthesis of starch. J Chem. Soc., 3692 6. Whelan, W. J. (1976) On the origin of pnimer for glycogen synthesis. Trends Biochem. Sci. 1, 13-15 7. Whelan, W. J. (1986) The initiation of glycogen synthesis. BioEssays 5, 136-140 8. Gieowar-Singh, D., Lomako, J., and Whelan, W. J. (1992) Purification of a seif-glucosylating protein from sweet corn. FASEBJ 6, A1520 (abstr.) 9. Krisman, C. R., and Barengo, R. (1975) A precursor of glycogen biosynthesis: alpha-i, 4-glucan-protein. Eur. j Biochem. 52, 117-123 10. Kennedy, L. D., Kirkman, B. R., Lomako, J., Rodriguez, I. R., and Whelan, W. J. (1985) The biogenesis of rabbit-muscle glycogen. In Membranes and Muscle (Berman, M. D., Gevers, W., and Opie, L. H., eds) pp. 65-84, IRL Press, Oxford 11. Rodriguez, I. R., and Whelan, W. J. (1985) A novel glycosylamino acid-linkage: rabbit-muscle glycogen is covalently linked
12.
13.
14.
15.
16.
17.
18.
19.
20.
to a protein via tyrosine. Biochem. Biophys. Res. Commun. 132, 829-836 Smythe, C., Caudwell, F C., Ferguson, M., and Cohen, P. (1988) Isolation and structural analysis of a peptide containing the novel tynosyl-glucose linkage in glycogenin. EMBO j 7, 2681-2686 Lomako, J., Lomako, W. M., and Whelan, W. J. (1992) The substrate specificity of isoamylase and the preparation of apoglycogenin. Carb. Res. 227, 331-338 Lomako, J., Lomako, W. M., and Whelan, W. J. (1988) A selfglucosylating protein is the primer for rabbit muscle glycogen biosynthesis. FASEBJ. 2, 3097-3103 Pitcher, J., Smythe, C., and Cohen, P. (1988) Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle. Eur. J. Biochem. 176, 391-395 Lomako, J., Lomako, W. M., and Whelan, W. J. (1991) Proglycogen: a low molecular-weight form of muscle glycogen. FEBS Lett. 279, 223-228 Judd, C., Lomako, J., Lomako, W. M., Ozdemir, Y., and Whelan, W. J. (1992) Proglycogen: an intermediate in glycogen synthesis. FASEBJ 6, A1520 (abstn.) Smythe, C., and Cohen, P. (1991) The discovery of glycogenin and the priming mechanism for glycogen biogenesis. Eur. j Biochem. 200, 625-631 Lomako,J., Lomako, W. M., and Whelan, W. J. (1990) The biogenesis of glycogen: nature of the carbohydrate in the protein primer. Biochem. Internat. 21, 251-260 Kulz, R. Z. (1886) Zur quantitativen Bestnimmung des Glykogens. Z BioL 22, 161-194
Vol. 6 October 1992 50 YEARS AGO 3219 w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 16, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
YEARSAGO something
This is the nineteenth in a series of articles recalling scien4fic events in FASEB 50 years ago, including reminiscences of the 1942 Annual Meeting.
On
the Initiation and Starch William
of Glycogen Synthesis J.
Whelan
It was exactly 50 years ago this month that I entered the University of Birmingham, U.K., as a 17-year-old undergraduate student, majoring in chemistry and supported by a scholarship from the Brewer’s Society, which wanted to bring more science into brewing. To that end, it was my ambition to become a biochemist, but wartime regulations prevented me from majoring in that subject. The plan that was put to me was to pursue chemistry, which was a large component of the biochemistry degree course (then called industrial fermentation), and perhaps by then the war would be over and I could make the switch. However, going through an accelerated curriculum, I graduated before the war ended, and after working for the Royal Navy on antisubmarine devices, I was able to pursue biochemical research in the same Department of Chemistry. The head of the Department was Sir Norman Haworth, the 1937 Nobel Laureate in Chemistry. He and his close associate, Stanley Peat, had gradually moved from strictly chemical to biochemical studies of starch, inspired by the work of Charles Hanes (1). In characterizing potato phosphorylase and carrying out the in vitro synthesis of starchlike material, Hanes in turn had received his inspiration from the work of Carl and Gerty Con and others, who had described mammalian phosphorylase and its ability to synthesize glycogen from glucose 1-phosphate (the Con ester) (2). I became interested in studying the mechanism of action of starch phosphorylase under the guidance of Peat, my thesis mentor. The connection of this account with the science of 1942 is that volume 147 of the Journal of Biological Chemistry contained two papers that were to be very important in my own researches. Arda Green and Con and Con (3) reported in a brief communication the crystallization of muscle phosphonylase and noted that for the crystalline enzyme to utilize glucose 1-phosphate in the synthesis of glycogen-like material,
there
was
the
same
dependency
on
added
glycogen
that they had reported earlier for less pure preparations of the enzyme (2). In other words, the synthesis of glycogen from glucose 1-phosphate depended on the addition of a trace of glycogen. The second paper was a full-length report by David Green and Paul Stumpf (4)-Green, in the unfamiliar role of describing the properties of starch phosphonylase, whereas we think of him for his enormous contributions over the next several decades in the field of bioenergetics. Green and Stumpf described many intriguing properties of potato phosphorylase and their paper became one that I consulted again and again. They showed that glycogen, starch, and dextrins derived therefrom were necessary to “catalyze” the polymerization of glucose from glucose 1-phosphate. They regarded the “catalyst” as literally that,
3218
that
accepted
phosphate
from
the Con
ester
and
then discharged it as inorganic phosphate. Con and Con (2) and Hanes (1) had referred to the phenomenon as “activation?’ The later concept that the role of the added carbohydrate in assisting starch (glycogen) synthesis was that of a primer, receiving glucose units from glucose 1-phosphate and becoming part of the finished macromolecule, had not been grasped, but had been by 1950 when we were able to show that of the oligosacchanide series based on glycogen and starch, maltotriose is the smallest that will accept glucose units in this way in the synthesis catalyzed by potato phosphorylase (5). The story of the search, through 1985, for the endogenous primer for glycogen and starch synthesis has been told elsewhere (6, 7). For glycogen, the details have emerged only in the last few years. For starch, the definitive answer is still not _______ at hand (8). The breakthrough came in the 1970s when Clara Krisman (9) reported her ability, with liver extracts, to synthesize glycogen-like material bound to protein. Following up this clue, we reported in 1985 the isolation of a covalently bound protein from muscle glycogen in one molecular proportion to each glycogen molecule. We named it glycogenin (10). The first alpha-glucose residue of glycogen, on which the synWilliam J. Whelan thesis of the molecule is initi-
________
ated, proved to be linked to glycogenin through a novel carbohydrate-to-protein bond, involving the hydroxyl group of a tyrosine residue (11-13). Next, glycogen-free glycogenin was isolated from a muscle extract and found to be an autocatalytic enzyme, creating a maltosaccharide primer on itself (14, 15). This does not direclty prime muscie-glycogen (macroglycogen, Mr lO7Da) synthesis, but that of a stable intermediate, proglycogen (Mr approx. 400 Da) (16), which functions both in the synthesis and degradation of macroglycogen (17). The more recent advances have been reviewed by Philip Cohen (18), whose laboratory has made important contributions, including the sequencing of glycogenin. Glycogen and starch were the first naturally occurring macromolecules to be synthesized in a cell-free system (1, 2), long before proteins or nucleic acids. But it was to be another 50 years before the nature of the initiation step for glycogen synthesis was understood. We had been correct, in our search for the endogenous primer, in assuming that it would be a maltosacchanide. We had been incorrect in looking for a system to synthesize a free oligosaccharide. Instead, it proved to be an autocatalytically synthesized, protein-bound maltosaccharide (malto-octaose, refs. 13, 19), which at the same time explained the reason for the occurrence of protein in glycogen, first reported 100 years ago (20). The answer to the nature of the primer was there all the time, inside the glycogen molecule, waiting to be uncovered.
Correspondence may be addressed to Dr. Whelan at the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, P.O. Box 016129, Miami, FL 33101, USA.
50 YEARS AGO
Vol. 6
October 1992
w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 16, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
REFERENCES 1. Hanes, C. 5. (1940) The reversible formation of starch from glucose-i-phosphate catalyzed by potato phosphorylase Proc. Roy. Soc. Lond. B129, 174-208 2. Con, G. T., and Con, C. F. (1940) The kinetics of the enzymatic synthesis of glycogen from glucose-l-phosphate. j BioL Chem. 135, 733-756 3. Green, A. A., Con, G. T., and Con, C. F (1940) Crystalline muscle phosphonylase. J BioL Chem. 142, 447-448 4. Green, D. E., and Stumpf, P. K. (1942) Starch phosphorylase of potato.]. BioL Chem. 142, 355-366 5. Bailey, J. M., Whelan, W. J., and Peat, S. (1950) Carbohydrate primers in the synthesis of starch. J Chem. Soc., 3692 6. Whelan, W. J. (1976) On the origin of pnimer for glycogen synthesis. Trends Biochem. Sci. 1, 13-15 7. Whelan, W. J. (1986) The initiation of glycogen synthesis. BioEssays 5, 136-140 8. Gieowar-Singh, D., Lomako, J., and Whelan, W. J. (1992) Purification of a seif-glucosylating protein from sweet corn. FASEBJ 6, A1520 (abstr.) 9. Krisman, C. R., and Barengo, R. (1975) A precursor of glycogen biosynthesis: alpha-i, 4-glucan-protein. Eur. j Biochem. 52, 117-123 10. Kennedy, L. D., Kirkman, B. R., Lomako, J., Rodriguez, I. R., and Whelan, W. J. (1985) The biogenesis of rabbit-muscle glycogen. In Membranes and Muscle (Berman, M. D., Gevers, W., and Opie, L. H., eds) pp. 65-84, IRL Press, Oxford 11. Rodriguez, I. R., and Whelan, W. J. (1985) A novel glycosylamino acid-linkage: rabbit-muscle glycogen is covalently linked
12.
13.
14.
15.
16.
17.
18.
19.
20.
to a protein via tyrosine. Biochem. Biophys. Res. Commun. 132, 829-836 Smythe, C., Caudwell, F C., Ferguson, M., and Cohen, P. (1988) Isolation and structural analysis of a peptide containing the novel tynosyl-glucose linkage in glycogenin. EMBO j 7, 2681-2686 Lomako, J., Lomako, W. M., and Whelan, W. J. (1992) The substrate specificity of isoamylase and the preparation of apoglycogenin. Carb. Res. 227, 331-338 Lomako, J., Lomako, W. M., and Whelan, W. J. (1988) A selfglucosylating protein is the primer for rabbit muscle glycogen biosynthesis. FASEBJ. 2, 3097-3103 Pitcher, J., Smythe, C., and Cohen, P. (1988) Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle. Eur. J. Biochem. 176, 391-395 Lomako, J., Lomako, W. M., and Whelan, W. J. (1991) Proglycogen: a low molecular-weight form of muscle glycogen. FEBS Lett. 279, 223-228 Judd, C., Lomako, J., Lomako, W. M., Ozdemir, Y., and Whelan, W. J. (1992) Proglycogen: an intermediate in glycogen synthesis. FASEBJ 6, A1520 (abstn.) Smythe, C., and Cohen, P. (1991) The discovery of glycogenin and the priming mechanism for glycogen biogenesis. Eur. j Biochem. 200, 625-631 Lomako,J., Lomako, W. M., and Whelan, W. J. (1990) The biogenesis of glycogen: nature of the carbohydrate in the protein primer. Biochem. Internat. 21, 251-260 Kulz, R. Z. (1886) Zur quantitativen Bestnimmung des Glykogens. Z BioL 22, 161-194
Vol. 6 October 1992 50 YEARS AGO 3219 w.fasebj.org by Iowa State University Serials Acquisitions Dept (129.186.138.35) on January 16, 2019. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
