Features of Glycogen Phosphorylase from the Body Wall Musculature of the LugwormArenicola marinaand the Mode of Activation during Anoxia.

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Abstract
The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 .+-. 1.2 nmol .times. min-1 .times. (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 .+-. 0.5 .mu.mol .times. min-1 .times. (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5''-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100mM. At 10mM phosphate the a-form could be activated 5-fold by AMP and showed a 40-fold higher affinity for AMP (KDiss = 20.mu.M) than the b form at 100mM phosphate. Caffeine (5 mM) inhibited completely the b form in presence of 1 mM AMP but not the a form and was therefore used to discriminate both forms. The findings suggest that glycogenolysis in the body wall musculature of Arenicola marina is not activated by phosphorylation during anoxia. The a form, however, which is already present under normoxic conditions might be activated by increasing levels of phosphate and AMP in the physiological concentration range, whereas the b form maintains virtually inactive.