Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens

Lederer, B; Stalmans, Willy

doi:10.1042/bj1590689

Cited by 34 publications

(17 citation statements)

References 17 publications

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“…The stimulation by AMP for the human enzyme (ϳ1.9-fold) was similar to that for the rat isozyme. The results are similar to those reported for human liver phosphorylase a from an autopsy specimen 5 h after death (39). In the latter study, AMP reduced the K m for glucose 1-phosphate by 50%.…”

Section: Discussionsupporting

confidence: 80%

“…In our laboratory, the specific activity of human liver phosphorylase a in the direction of glycogen synthesis was previously reported to be 40 U/mg of protein in liver biopsy specimens from organ donors (69), i.e., similar to that for the recombinant human enzyme. In an autopsy specimen (39), others have reported it to be only 14.4 U/mg protein (39). This low activity may be attributable to the tissue being obtained 5 h after death (69).…”

Section: Discussionmentioning

confidence: 97%

“…Similarly, AMP has strikingly different effects on the saturation kinetics of human and rat liver phosphorylase b enzymes, even though the AMP-binding site is highly conserved (39,62). AMP increases the maximum velocity (V max ) of the human enzyme 10-fold without affecting the affinity for the substrate (K m ).…”

Section: Discussionmentioning

confidence: 99%

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Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Ercan-Fang

Gannon

Rath

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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Hepatic glucose production is increased in people with type 2 diabetes. Glucose released from storage in liver glycogen by phosphorylase accounts for ϳ50% of the glucose produced after an overnight fast. Therefore, understanding how glycogenolysis in the liver is regulated is of great importance. Toward this goal, we have determined the kinetic characteristics of recombinant human liver glycogen phosphorylase a (HLGPa) (active form) and compared them with those of the purified rat enzyme (RLGPa). The Michaelis-Menten constant (K m) of HLGPa for Pi, 5 mM, was about fivefold greater than the Km of RLGPa. Two Pi (substrate) concentrations were used (1 and 5 mM) to cover the physiological range for P i. Other effectors were added at estimated intracellular concentrations. When added individually, AMP stimulated, whereas ADP, ATP and glucose inhibited, activity. These results were similar to those of the RLGPa. However, glucose inhibition was about twofold more potent with the human enzyme. UDP-glucose, glucose 6-phosphate, and fructose 1-phosphate were only minor inhibitors of both enzymes. We reported previously that when all known effectors were present in combination at physiological concentrations, the net effect was no change in RLGPa activity. However, the same combination reduced HLGPa activity, and the inhibition was glucose dependent. We conclude that a combination of the known effectors of phosphorylase a activity, when present at estimated intracellular concentrations, is inhibitory. Of these effectors, only glucose changes greatly in vivo. Thus it may be the major regulator of HLGPa activity. glucose; glycogen metabolism; enzyme regulation; adenosine 5Ј-monophosphate; adenosine 5Ј-diphosphate; adenosine 5Ј-triphosphate; glucose 1-phosphate; fructose 1-phosphate; uridine 5Ј-diphosphate-glucose IN HUMANS, GLUCOSE RELEASED from liver glycogen by the enzyme phosphorylase accounts for ϳ50% of the total glucose produced after an overnight fast, significantly contributing to the maintenance of a normal blood glucose concentration (11,27,38,53,63). Despite increased serum glucose level in people with type 2 diabetes, phosphorylase-mediated glycogenolysis continues to contribute ϳ40-50% of overnight glucose production, inappropriately maintaining hyperglycemia (3,4,21,63, 68). Once glycogenolysis decreases, as with extended fasting, the blood glucose concentration decreases dramatically (3,20,24,32, 68). Therefore, understanding how phosphorylase-mediated glycogenolysis is regulated in people with or without diabetes is of considerable importance.Phosphorylase removes glycosyl units from the terminal branches of glycogen through phosphorolysis, forming glucose 1-phosphate. It is present in two interconvertible forms, phosphorylase a (phosphorylated) and b (unphosphorylated). The a form is the active form. Regulation of phosphorylase is incompletely understood. Phosphorylase has been postulated to be primarily regulated by the interconversion of the active, phosphorylated (phosphorylase a) and inactive, nonphospho...

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 97%

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Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Ercan-Fang

Gannon

Rath

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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“…This report provides evidence that the answer is positive. Some of the results described here have been presented before under a preliminary form [7].…”

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confidence: 96%

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

Lederer

1978

European Journal of Biochemistry

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Baker's yeast L-lactate dehydrogenase (flavocytochrome b2) is a typical flavodehydrogenase, in that it accepts two electrons from the substrate but has a monoelectronic acceptor. Yet it forms a red semiquinone [Capeillere Blandin et al. Eur. J. Bioclwm. 54, 549-566 (1975)j and it is shown in this paper that it forms a reversible covalent complex with sulfite (Kd = 1.4 pM). This complex can be observed by difference spectroscopy and provides a convenient tool for visualizing the flavin chromophore, usually hidden behind the intense heme absorbance. A number of anions (D-lactate, oxalate and pyruvate) are inhibitors of the enzymatic reaction and induce spectral perturbations of the flavin spectrum. It is concluded that probably two positive charges exist at the active site: one which stabilizes the red semiquinone and one which attracts organic anions and sulfite. It is also concluded that the correlation between reactivity with sulfite and reactivity with oxygen among flavoproteins may not be as general as previously proposed [Massey et al. J. Biol. Chem. 244,[3999][4000][4001][4002][4003][4004][4005][4006] (1 969)].Free reduced flavin is rapidly reoxidized by oxygen, whereas enzyme-bound flavin either keeps or loses this property, according to the function of the protein. This phenomenon provides the basis for the now classical distinction, among flavoproteins, between oxidases and dehydrogenases. It is clear that the protein moiety plays a fundamental role in modulating the reactivity of the bound flavin, but this role is at present poorly understood, in spite of numerous efforts.A few years ago, Massey et al. [l] proposed an interesting correlation between the reactivity of flavoproteins toward oxygen and toward sulfite. It was shown that a number of flavoproteins were bleached by sulfite, which reversibly formed a covalent bond with N-5 of the flavin nucleus [2]; those proteins that reacted were oxidases and formed anionic (red) semiquinones; those that did not react were dehydrogenases and formed neutral (blue) semiquinones. A tentative explanation was presented, based on these and other observations: a positive charge at the active site of oxidases would facilitate sulfite binding and stabilize the anionic semiquinone ;

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“…8 Phosphorylase was measured by determination of inorganic phosphate released from 0.1 M G-1-phosphate in the presence of 1% glycogen and 2 mM adenosine monophosphate. 15 …”

Section: Enzyme and Glycogen Analysismentioning

confidence: 99%

Canine Model and Genomic Structural Organization of Glycogen Storage Disease Type Ia (GSD Ia)

et al. 2001

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Abstract. A canine model of glycogen storage disease Ia (GSD Ia), similar clinically, biochemically, and pathologically to the human disease, was established by crossbreeding Maltese and Beagle dogs carrying a mutated, defective glucose-6-phosphatase (G-6-Pase) gene. Ten puppies were born in three litters from these crossbreedings. Six were homozygous for the previously described M121I GSD Ia mutation. Of these six affecteds, two were stillborn, and one died at 2, 32, and 60 days of life, respectively (puppies A, B, C, D, E), while one is alive at age 15 months (puppy F). Affected puppies exhibited tremors, weakness, and neurologic signs when hypoglycemic. They had postnatal growth retardation and progressive hepatomegaly. Biochemical abnormalities included fasting hypoglycemia, hyperlactacidemia, hypercholesterolemia, hypertriglyceridemia, and hyperuricemia. Microscopic examination of tissues from affected puppies showed diffuse, marked hepatocellular vacuolation, with distended clear hepatocytes and central to marginally located rounded nuclei. In the kidneys of puppies D and E, there was segmental glomerular sclerosis and vacuolation of proximal convoluted tubular epithelium. Biochemical analysis revealed increased liver glycogen content and isolated markedly reduced G-6-Pase enzyme activity in liver and kidney. The canine G-6-Pase gene was characterized by screening a canine genomic library. It spans ϳ11.8 kb and consists of five exons with Ͼ90% amino acid sequence homology to the derived human sequence. The first 1.5 kb of the 5Ј region was sequenced and contains several putative response element motifs homologous to the human 5Ј region. Establishment of this canine colony of GSD Ia that closely resembles human disease and isolation of the canine genomic gene provides an excellent model for studying pathophysiology and long-term complications and an opportunity to develop novel therapeutic approaches such as drug and gene therapy.

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Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens

Cited by 34 publications

References 17 publications

Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast

Canine Model and Genomic Structural Organization of Glycogen Storage Disease Type Ia (GSD Ia)

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Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens

Cited by 34 publications

References 17 publications

Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Integrated effects of multiple modulators on human liver glycogen phosphorylasea

Sulfite Binding to a Flavodehydrogenase, Cytochrome b2 from Baker's Yeast

Canine Model and Genomic Structural Organization of Glycogen Storage Disease Type Ia (GSD Ia)

Contact Info

Product

Resources

About

Sulfite Binding to a Flavodehydrogenase, Cytochrome b₂ from Baker's Yeast