Muscle phosphorylase absence

Muscle phosphorylase is distinct from that of Hver. It is a dimer, each monomer containing 1 mol of pyridoxal phosphate (vitamin Bg). It is present in two forms phos-phoiylase a, which is phosphorylated and active in either the presence or absence of 5 -AMP (its allosteric modifier) and phosphorylase h, which is dephosphorylated and active only in the presence of 5 -AMP. This occurs during exercise when the level of 5 -AMP rises, providing, by this mechanism, fuel for the muscle. Phosphorylase a is the normal physiologically active form of the enzyme. 

TypeV Myophosphorylase deficiency, McArdle s syndrome Absence of muscle phosphorylase Diminished exercise tolerance muscles have abnormally high glycogen content (2.5-4.1%). Little or no lactate in blood after exercise. 

Fig. 8. The activity of muscle phosphorylase b (as a logarithm percentage of the activity of the fully AMP-activated enzyme in water) as a funaion of concentration (% v/v) of added organic cosolvent in the absence of AMP. It can be seen that long

Fig. 7.18. Regulation of glycogen metabolism in muscle. Phosphorylase kinase stands at the center of regulation of glycogen metabolism. Phosphorylase kinase may exist in an active, phosphorylated form and an inactive, unphosphorylated form. Phosphorylation of phosphorylase kinase is triggered by hormonal signals (e.g. adrenahne) and takes place via an activation of protein kinase A in the cAMP pathway. In the absence of hormonal stimulation, phosphorylase kinase can also be activated by an increase in cytosolic Ca. The active phosphorylase kinase stimulates glycogen degradation and inhibits glycogen synthesis, in that, on the one side, it activates glycogen phosphorylase by phosphorylation, and on the other side, it inactivates glycogen synthase by phosphorylation.

The phosphorylase-stimulation assay247 250 is based on the stimulation by branching enzyme of the unprimed reaction in absence of primer activity seen with rabbit muscle phosphorylase a activity. Branching enzyme present in the reaction mixture increases the number of non-reducing ends available to phosphorylase for elongation. 

Other ions, or molecules, may be required for maximal activity of an enzyme, although the enzyme is active in their absence. Thus, mammalian alpha-amylases are activated by chloride ions, and rabbit-muscle phosphorylase a is activated by adenosine 5-phosphate. 

In the Type VI diseases, there appears to be abnormally low or complete absence of phosphorylase activity. In three cases (Type Via) examined by Hers, the activity of the liver phosphorylase was about 5 units, compared with values of 12-33 units for other cases of glycogen-storage disease, although the muscle phosphorylase activity was normal. 

This is due to the absence of muscle phosphorylase, severely limiting glycogen degradation and the capacity for sustained strenuous exercise, because muscle glycogen is unavailable as an every source. There usually is muscle damage accompanying this disease. 

The changes in the quaternary structure of rabbit-muscle phosphorylase b at 4 °C have been followed by measuring the reactivity of the thiol group. A slow, concerted transition between the two dimeric forms was shown to take place, the rate of which is affected by adenosine monophosphate. A phos-phopeptide containing 14 residues, including the phosphorylated serine residue, derived from the amino-terminus of rabbit skeletal-muscle phosphorylase has been shown to induce the enzymic properties of phosphorylase a in phosphorylases b and b (a modified form in which the phosphorylated site has been removed by limited digestion with trypsin). Thus, these enzymes become partially active in the absence of adenosine monophosphate. 

This is due to the absence of muscle phosphorylase activity. Phos-phorylase is one of the glycolytic enzymes. 

In resting muscle, nearly all the enzyme is in the inactive b form. When exercise commences, the elevated level of AMP leads to the activation of phosphorylase b. Exercise will also result in hormone release that generates the phosphorylated a form of the enzyme. The absence of glucose 6-phosphatase in muscle ensures that glucose 6-phosphate derived from glycogen remains within the cell for energy transformation. 

The enzyme from rabbit muscle may be extracted and crystallized readily, but many recrystallizations may be necessary to ensure freedom from such contaminants as alpha-amylase, debranching enzyme, and branching enzyme. Purification may be facilitated by the use of column chromatography on 0-(2-diethylaminoethyl) cellulose. The enzyme exists in two forms (a and h) that differ in their requirement for adenosine 5 -monophosphate as cofactor. The b form is inactive in the absence of cofactor, and may be converted into the active (adenosine 5 -monophos-phate-independent) a form by phosphorylation of a specific serine residue under the action of (contaminant-free) phosphorylase kinase with adenosine 5 -triphosphate as the phosphate donor. 

If the phosphorylase pool plays an important part in vitamin B6 kinetics, it might be anticipated that this metabolism would be disturbed in patients suffering from MciVdle s disease, a rare metabolic myopathy caused by an absence of functional muscle glycogen phosphorylase. The absence of this enzyme means that patients cannot break down their muscle glycogen reserves. Other energy sources within the muscle are rapidly depleted... 

There are a number of steps in the expression of a functional protein from a gene and a disruption at any point can halt the normal expression of the protein. Consequently, there is variation between patients in their expression of phosphorylase mRNA and protein (McConchie et al., 1991). The majority of individuals do not express protein or mRNA, which is due either to a failure to transcribe the gene or due to accelerated degradation of the transcript. A smaller number of patients do express a stable transcript but it is not subsequently translated into protein and even fewer patients express protein but at low levels (approximately 1% of normal values). The majority of McArdle s patients are characterized by a complete absence of the protein in muscle, consistent with knowledge of the mutations in McArdle s disease. 

The phosphorylases are everywhere present in the biosphere. An enz5rme of this type (phosphorylase-a) has been obtained crystalline from rabbit muscle. In the presence of an excess of orthophosphate the reaction occurs in the direction of phosphorolysis, whilst in the absence of orthophosphate or in the presence of an excess of glucose-l-phosphate, the synthesis of amylose takes place. The enzyme is also called glucose-l-phosphate amylose-transglucosidase. 

Raising intracellular levels of cyclic AMP stimulates lipolysis in fat pads and isolated cells (4) probably by activating the triglyceride lipase. The nucleotide also stimulates the conversion of phosphorylase b to phosphorylase in muscle and the conversion of the I form of glycogen synthetase to the D form (5, 6). It thus seemed possible that the inhibitory action of insulin on these processes might be mediated by a fall in tissue levels of cyclic AMP. Accordingly we have examined the effect of insulin on cyclic AMP levels in epididymal fat pads incubated with epinephrine and caffeine in the absence of glucose. 

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