Phosphofructokinase from Ascaris suum. Purification and properties.

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.

Glycogen metabolizing enzymes during starvation and feeding of Ascaris suum maintained in a perfusion chamber.

The glycogen content of muscle was correlated with the activity of glycogen synthase and glycogen phosphorylase from the parasitic roundworm Ascaris suum maintained in vitro. Adult female worms were maintained in the laboratory in a perfusion system during periods of starvation and feeding. During starvation, the levels of glucogen decreased at a rate of 0.1 to 0.2 mumoles/min/g wet weight of muscle-cuticle. During this time, 95% of the glycogen synthase (E.C. 2.4.1.11) was in the active D-form, and 48% of the phosphorylase (E.C. 2.4.1.1) was in the active a-form. Upon feeding, the rate of incorporation of glycosyl residues into glycogen proceeded at a rate of 0.75 to 1.0 mumoles/min/g muscle-cuticle. Glycogen synthase was 22% in the active I-form and phosphorylase a-levels remained virtually unchanged at 41% as compared with the starved worm. Total levels of both enzymes remained constant over the starvation-feeding period with 3.9 units/g phosphorylase and 0.4 units/g glycogen synthase. The apparent Km value for the substrate UDPG for glycogen synthase was 0.22 +/- 0.02 mM. For glycogen phosphorylase the Km value for G-1-P was 1.76 +/- 0.38 mM.

Activity of enzymes regulating glycogen metabolism in perfused muscle-cuticle sections of Ascaris suum (Nematoda).

A new perfusion system has been developed in which muscle-cuticle sections of Ascaris suum were perfused, enabling study of enzymes in vitro. Using this technique the activity of the regulatory enzymes glycogen synthase and glycogen phosphorylase was determined, and the level of glycogen in the muscle was assessed. During starvation, 98% of glycogen synthase was in the inactive D-form, and 80% of the glycogen phosphorylase activity was in the active a-form. When the ascarid muscle section was perfused with 27 mM glucose, 13.1% of the glycogen synthase was in the active I-form, whereas phosphorylase a-levels dropped to 46% and glycogen was synthesized at a linear rate of 12 mg/g/hr or 1.23 mumoles/min/g muscle-cuticle. ATP levels (3.71 +/- 0.32 mM) remained unchanged over a 4-hr perfusion period with an adenylate energy charge of 0.82. Fructose supported glycogen synthesis, though not as well as glucose. Galactose, mannose, and trehalose did not support glycogen synthesis. The new perfusion system should be useful in future, similar studies on Ascaris.