Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.

The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.

Purification and properties of cyclic-3',5'-GMP-dependent protein kinase from the nematode Ascaris suum.

A cyclic-3',5'-GMP-dependent protein kinase was purified 7400-fold from the reproductive tract of female ascarids to a specific activity of 718 nmol min-1 mg-1 (histone as substrate). The yield of the preparation was 25%. The enzyme protein obtained was homogeneous as judged by isoelectrofocusing and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The native enzyme behaved as a dimer of two 82-kDa subunits in gel permeation chromatography on Superose 12. The protein kinase was inactive in the absence of cyclic purine nucleotides. Half-maximum velocity was obtained in the presence of 18 nM cGMP, whereas 400-fold higher concentrations of cAMP were required for the same activity. The enzyme underwent autophosphorylation in first-order kinetics (rate constant 0.054 min-1), leading to maximum incorporation of 0.96 phosphate per subunit. The autophosphorylation led to a 4-fold increase in Vmax, while the Km remained almost unchanged. In an extract from the reproductive tract, cGMP-stimulated phosphorylation was primarily observed in five proteins (molecular masses of 66, 60, 43, 30, and 25 kDa). These proteins also incorporated phosphate when isolated reproductive tracts were incubated in the presence of [32P]phosphate. The phosphate content in cellular proteins was enhanced when the incubation was performed in the presence of 10(-4) M of either octyl-cAMP or octyl-cGMP. In addition to the proteins mentioned above, however, six more electrophoretic bands containing radioactive phosphate were identified after in situ labeling of reproductive tracts with radioactive phosphate.

Purification and properties of the cyclic 3',5'-AMP-binding protein from the muscle of the Nematode Ascaris suum.

The cyclic 3',5'-AMP-binding protein was isolated from the muscle of Ascaris suum and purified to apparent homogeneity. It migrated as a protein with a relative Mr 54,000 on electrophoresis under denaturing conditions. On gel filtration columns it was eluted at a volume corresponding to a protein of Mr greater than 200,000 under conditions which kept the cyclic 3',5'-AMP-binding property intact. The purified catalytic subunit of protein kinase from Ascaris and the C subunit of cyclic 3',5'-AMP-dependent protein kinase from bovine heart were inhibited by the cyclic 3',5'-AMP-binding protein. Gel filtration studies indicated the formation of a stable protein complex between the protein kinase and the cyclic 3',5'-AMP-binding protein from Ascaris.

Identification of two different phosphofructokinase-phosphorylating protein kinases from Ascaris suum muscle.

Two different phosphofructokinase-phosphorylating protein kinases were separated from extracts of Ascaris suum muscle by chromatography on DEAE-Fractogel. They were tentatively designated phosphofructokinase kinase I and phosphofructokinase kinase II. Phosphofructokinase kinase I eluted from the chromatography column at an ionic strength of 0.07 and contained about 25% of the phosphofructokinase-phosphorylating activity assayed in crude extracts. The protein kinase activity was not stimulated by the addition of either cAMP or cGMP. It was inhibited by the heat-stable protein kinase inhibitory protein from rabbit muscle (Walsh inhibitor), by the regulatory subunit of cAMP-dependent protein kinase from beef heart, and by the cAMP-binding protein from Ascaris muscle. These properties suggest that phosphofructokinase kinase I is homologous to the catalytic subunit of cAMP-dependent protein kinases from mammals. This assumption is supported by the estimation of the Mr of 40,000 for the purified phosphofructokinase kinase I under denaturing conditions and by the fact that the presence of cAMP eliminated the inhibition by the cAMP binding proteins. The isoelectric point of the enzyme was 8.7. Phosphofructokinase kinase II was eluted from the DEAE-Fractogel column at an ionic strength of 0.16 and contained approximately 75% of the phosphofructokinase kinase activity measured in the extracts. The molecular and kinetic properties were significantly different from those of phosphofructokinase kinase I. The enzyme was not inhibited by the heat-stable inhibitor protein nor by cAMP-binding proteins. The Mr of the native enzyme was estimated as 220,000 by molecular sieve chromatography. The isoelectric point of the enzyme was pH 5.45.

Reversible activation and inactivation of phosphofructokinase from Ascaris suum by the action of tissue-homologous protein phosphorylating and dephosphorylating enzymes.

In the presence of ATP-Mg2+, purified phosphofructokinase from Ascaris suum muscle was effectively phosphorylated and activated in vitro by a protein kinase purified from the same tissue. Both effects were reversed by the action of a purified protein phosphatase from the same tissue. The findings suggest the presence of a highly potent interconversion mechanism for phosphofructokinase in the muscle of the parasitic nematode.