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Glycogen phosphorylase and its converter enzymes in haemolysates of normal human subjects and of patients with type VI glycogen-storage disease. A study of phosphorylase kinase deficiency.

ABSTRACT: 1. The properties of phosphorylase a, phosphorylase b, phosphorylase kinase and phosphorylase phosphatase present in a human haemolysate were investigated. The two forms of phosphorylase have the same affinity for glucose 1-phosphate but greatly differ in Vmax. Phosphorylase b is only partially stimulated by AMP, since, in the presence of the nucleotide, it is about tenfold less active than phosphorylase a. In a fresh human haemolysate phosphorylase is mostly in the b form; it is converted into phosphorylase a by incubation at 20degreesC, and this reaction is stimulated by glycogen and cyclic AMP. Once activated, the enzyme can be inactivated after filtration of the haemolysate on Sephadex G-25. This inactivation is stimulated by caffeine and glucose and inhibited by AMP and fluoride. The phosphorylase kinase present in the haemolysate can also be measured by the rate of activation of added muscle phosphorylase b, on addition of ATP and Mg2+. 2. The activity of phosphorylase kinase was measured in haemolysates obtained from a series of patients who had been classified as suffering from type VI glycogenosis. In nine patients, all boys, an almost complete deficiency of phosphorylase kinase was observed in the haemolysate and, when it could be assayed, in the liver. A residual activity, about 20% of normal, was found in the leucocyte fraction, whereas the enzyme activity was normal in the muscle. These patients suffer from the sex-linked phosphorylase kinase deficiency previously described by others. Two pairs of siblings, each time brother and sister, displayed a partial deficiency of phosphorylase kinase in the haemolysate and leucocytes and an almost complete deficiency in the liver. This is considered as being the autosomal form of phosphorylase kinase deficiency. Other patients were characterized by a low activity of total (a+b) phosphorylase and a normal or high activity of phosphorylase kinase in their haemolysate.

SUBMITTER: Lederer B

PROVIDER: S-EPMC1165371 | biostudies-other | 1975 Apr

REPOSITORIES: biostudies-other

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Project description:In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. Pru?cdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the ?cdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced brain cysts in infected mice, but the numbers of cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored brain cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the brain.

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Glycogen phosphorylase and its converter enzymes in haemolysates of normal human subjects and of patients with type VI glycogen-storage disease. A study of phosphorylase kinase deficiency.

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