Phosphorylase turnover

III. Labeling Methods to Monitor Phosphorylase Turnover A. Radiolabelled Cofactor... 

Our interest in phosphorylase turnover has led us to appreciate the importance of the phosphorylase-bound pool of vitamin B6. The behavior... 

Experiments were also carried out injecting [3 -3H]xylosyl-MTA. The results indicated that the molecule has a very low turnover rate in D. verrucosa, since 96% of the recovered radioactivity after 24 h was associated with xylosyl-MTA. Accordingly, it was observed [126] that xylosyl-MTA is resistant to the enzyme MTA-phosphorylase which cleaves MTA but not the xylose analog, which therefore accumulates in the animal. Since xylosyl-MTA is mainly concentrated in the hermaphrodite gland of D. verrucosa and is very abundant in the eggmasses [103], it may play a role in the reproductive biology of D. verrucosa. 

There may be multiple metabolicpoolsofthe vitamin, with very different rates of turnover. In this case, short-term and long-term smdies will give very different estimates of the fractional rate of turnover of the total body pool. As discussed in Section 9.6.1, this is known to be a problem with vitamin Be, because some 80% of the total body pool is associated with muscle glycogen phosphorylase and has a much lower fractional turnover rate than the remaining 20%. 

This is considerably lower than the requirements estimated from depletion/ repletion studies (Section 9.6.2) and may reflect dilution of the small pool associated with amino acid metaholism, which has a rapid turnover, by the larger and more stable pool associated with glycogen phosphorylase. 

This large, slow turnover pool is most likely in muscle, which contains about 70-80% of the vitamin B6 in the body (Cobum et al, 1985,1988b). The muscle pool is associated primarily with glycogen phosphorylase (Butler et al, 1985). Using pyridoxal phosphate as an indicator of glycogen phospho-... 

A third consequence is the question of whether glycogen phosphorylase conserves vitamin B6 because it has a very low rate of degradation or because it recycles the pyridoxal phosphate very efficiently. Based on the rapid decay of the free vitamin B6 pool in muscle, the slow turnover of the protein-bound vitamin B6 pool, the failure to detect apo-phosphorylase, and the agreement between turnover rates based on amino acid or vitamin B6 data, Beynon et al. (1986) concluded that recycling would be minimal. We feel that the effect of vitamin B6 intake on turnover rates tends to favor the recycling option under conditions of limited intake. However, more data are needed before a final decision can be made. 

Beynon, R. J., Fairhurst, D.. and Cookson, E. J. (1986). Turnover of skeletal muscle glycogen phosphorylase. Biomed. Biochim. Acta 45,1619-1625. 

Butler, P. E., Cookson, E. J., and Beynon, R. J. (1985). The turnover of skeletal musde glycogen phosphorylase studied using the cofactor, pyridoxal phosphate, as a specific label. Biochim. Biophys. AcU> 847, 316-323. 

Two properties of the muscle phosphorylase pool associated with PLP (its large size and slow kinetics of exchange) led us to speculate that the cofactor might provide a specific label with which to monitor the turnover of the enzyme in vivo (Fig. 1). Implicit in this suggestion is the requirement that the cofactor is incorporated into the enzyme as a cosynthetic or immediately postsynthetic event. Further, reutilization of cofactor within mus-... 

FIG. 1. Interrelationships between vitamin B6 and phosphorylase metabolism. The low rate of turnover of glycogen phosphorylase (gpb) and the lack of exchange of free and protein-bound PLP mean that exchange into the muscle pool is largely controlled by the kinetics of turnover of the enzyme. At present, it is not known whether resolution of the holo-enzyme is a prerequisite or consequence of phosphorylase degradation. Reproduced with permission of llie Biochemical Journal. 

Ten days after injection of label, the low-molecular-weight pool contained virtually no label, whereas label remained associated with phosphorylase. Subsequent isolation of phosphorylase from different animals over a range of time periods between 10 and 30 days defined an exponential decay, the rate constant of which was taken as the rate of degradation of phosphorylase. This was subsequently confirmed by independent measurement of the rate of turnover of the enzyme using continuous infusion of labeled amino acids—a method that is independent of reutilization artifacts. The rate of turnover of phosphorylase was the same when measured by either method (Beynon et al., 1986 Cookson and Beynon, 1989). 

Phosphorylase has a relatively low rate of turnover, and as such, large doses of radiolabeled pyridoxine were needed to obtain an adequate degree of labeling. In addition, the need for serial sampling of the decay curve, using individual animals, introduced substantial biological variation. Both aspects of this experimental system precluded application of the method to humans, and we considered the possibility of a different approach, based on stable-isotope-labeled pyridoxine, to monitor phosphorylase degradation. 

The kinetics of the rise to a plateau value can be analyzed by nonlinear curve fitting as a biexponential equation and yields rate constants for turnover of the large and slow components, from which the rate constant of loss of PLP from the phosphorylase pool (=rate of degradation of enzyme) can be calculated (Coburn, Chapter 6, this volume). 

The rate constant for turnover of the slow component was 0.13 0.03/ day (mean SEM., n = 10) from which a value of turnover of the phosphorylase pool can be calculated as 0.1/day (Beynon etal, 19%). This compares well with values of 0.12/day obtained for gastrocnemius muscle (Leyland et ai, 1990) and 0.13/day for total hind limb and back muscle (Leyland and Beynon, ). The fast pool (presumed to be all labile forms of the vitamin) was turning over very quickly, with a rate constant of 1.3 0.4/day (a half-life of 12 hr). However, the experimental protocol that we use does not permit acquisition of a sufficiently detailed data set to acquire accurate kinetics on the fast pool. This preliminary analysis of the data also implies that the fast pool accounts for about 50% of the total vitamin B6 in the body—it is not yet clear whether this is consequential to the inability to define the fast phase with a high degree of precision or whether the muscle phosphorylase itself partitions into two pools that differ in accessibility. For example, enzyme bound to the glycogen particle might be more stable than enzyme free in the sarcoplasm. Further work is needed to resolve these issues. 

Beynon, R. J., leyland, D. M., Evershed, R. P., Edwards, R. H. T., and Cobum, S. P. (1996). Measurement of the turnover of glycogen phosphorylase by gas chromatography/mass spectrometry using stable isotope derivatives of pyridoxine (Vitamin B6). Biochem. J. 317, 613-619. 

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