Effect of reductive alkylation on catalytic properties of glycogen phosphorylase B. enzyme derivatives with changed nucleotide affinities.

Glycogen phosphorylase b modified by NaBH4 and aliphatic aldehydes of varying chain length: ranging from acetaldehyde to n-heptanaldehyde were purified by heat-treatment. Kinetic studies showed that the various purified enzyme derivatives were similar to native phosphorylase b in kinetic properties with respect to glucose-I-P. They, however, exhibited different affinities toward AMP. For acetaldehyde, propionaldehyde and butyraldehyde modified phosphorylases b, increase in chain length of the modifying aldehyde resulted in a decrease in AMP affinity of the modified enzyme. For the other aldehydes increase in chain length resulted in an increased AMP affinity of the modified enzyme. Consequently, the apparent values of butyraldehyde and heptanaldehyde modified phosphorylase b exhibited greatest deviation from that of native phosphorylase b. These two enzyme derivatives were studied in more detail. The activation of both enzyme derivatives by AMP could be greatly stimulated by spermine at suboptimal levels of AMP. Both derivatives could be activated by IMP, UMP or CMP in addition to AMP. The extents of activation of heptanaldehyde modified phosphorylase b by these nucleotides were higher than those found for native phosphorylase b. Thus, under certain conditions which included using suboptimal AMP or using IMP, UMP or GMP instead of AMP, heptanaldehyde modified phosphorylase b had much higher catalytic activity than native phosphorylase b. The interactions between burtyraldehyde or heptanaldehyde modified phosphorylases b with IMP were respectively weaker or stronger than that between native phosphorylase b and IMP. The interactions between butyraldehyde or heptanaldehyde modified phosphorylase b with glucose-6-P, an inhibitor of phosphorylase b partially competitive with respective to AMP, were also respectively weaker or stronger than that between the inhibitor and the native enzyme.

Inhibition of Ca2+-activated cyclic nucleotide phosphodiesterase reaction by a heat-stable inhibitor protein from bovine brain.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.

Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.