The differential effects of the oxyanions of the early transition metals ions V(V), W(VI), and Mo(VI) on the catalytic activity and coenzyme binding of rabbit skeletal muscle phosphorylase are studied. The oligoanions of V(V), W(VI), and Mo(VI) are potent inhibitors of phosphorylase. Kinetic studies revealed that oligovanadates inhibit pyridoxal-reconstituted phosphorylase b by competing with both the substrate, glucose 1-phosphate, and the activator, phosphite, with K1 values of 4 microM and 6 microM, respectively. Oligovanadates in the millimolar concentration range inhibit phosphorylases a and b by competing with glucose 1-phosphate binding. The polymeric decavanadate and paratungstates caused time-dependent inactivation of phosphorylase. Spectral studies with tungstate and phosphorylase b revealed that the inactivation is due to deformation of the coenzyme site. Kinetic studies and the protective effects of substrate and effectors on inactivation and deformation by tungstate or vanadate suggest that deformation and inactivation is caused by a primary binding of the oligoanions at the glucose 1-phosphate site. Nuclear magnetic resonance (NMR) studies of vanadate-phosphorylase complexes and vanadate solutions under different conditions were carried out to ascertain the nature of vanadate ions interacting with functional groups in phosphorylase. The results suggest that decavanadate is the major protein-bound species. NMR studies also showed that guanidino groups react with decavanadate and suggest that arginine residues in phosphorylase are potential functional groups that can interact with decavanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridoxal-reconstituted phosphorylase was used as a model system to study the possible functions of the 5'-phosphoryl group of pyridoxal 5'-phosphate (PLP) in rabbit muscle glycogen phosphorylase. Kinetic study was conducted by using competitive inhibitors of phosphite, an activator, and alpha-D-glucopyranose 1-phosphate (glucose-1-P) to study the relationship between the PLP phosphate and the binding of glucose-1-P to phosphorylase. Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy of fluorophosphate bound to pyridoxal phosphorylase showed that its ionization state did not change during enzymatic catalysis. Evaluation of the apparent kinetic parameters for the activation of pyridoxal phosphorylase with different analogues having varied pKa2 values demonstrated a dependency of KM on pKa2. Molybdate, capable of binding as chelates in a trigonal-bipyramidal configuration, was tested for its inhibitory property with pyridoxal phosphorylase. On the basis of the results in this study, several conclusions may be drawn: (1) The bound phosphite in pyridoxal phosphorylase and, possibly, the 5'-phosphoryl group of PLP in native phosphorylase do not effect the glucose-1-P binding. (2) One likely function of the 5'-phosphoryl group of PLP in native phosphorylase is acting as an anchoring point to hold the PLP molecule and/or various amino acid side chains in a proper orientation for effective catalysis. (3) The force between the PLP phosphate and its binding site in phosphorylase is mainly electrostatic; a change of ionization state during catalysis is unlikely. (4) Properties of the central atoms of different anions are important for their effects as either activators or inhibitors of pyridoxal phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)

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A protein with an apparent molecular size of 490 kDa was found in the postsynaptic density (PSD) fraction isolated from porcine cerebral cortices and rat forebrains, and this 490 kDa protein accounted for $3% of the total protein of these samples. Matrix-assisted laser desorption ionization-time of flight mass spectrometric and Western blotting analyses consistently indicated that this 490 kDa protein consisted primarily of the heavy chain of cytoplasmic dynein (cDHC). Immunocytochemical analyses showed that cDHC was found in 92% and 89% of the phalloidin-positive protrusions that were themselves associated with discrete clusters of synaptophysin, a presynaptic terminal marker, and PSD-95, a postsynaptic marker, on neuronal processes, respectively. Quantitative Western blotting analyses of various subcellular fractions isolated from porcine cerebral cortices and rat forebrains further showed that not only the heavy but also the intermediate chains of dynein are enriched in the PSD fraction. Cytoplasmic dynein is a microtubule-associated motor protein complex that drives the movement of various cargos toward the minus ends of microtubules and plays many other diverse functions in the cell. Our results that cDHC is a major component of the PSD fraction, that both dynein heavy and intermediate chains are enriched in the PSD fraction and that cDHC is present in dendritic spines raise the possibilities that cytoplasmic dynein may play structural and functional roles in the postsynaptic terminal. V V C 2006 Wiley-Liss, Inc.

A new vitamin B6 analogue, 6-fluoro-5'-deoxypyridoxal (6-FDPL), was synthesized and characterized. This analogue, as well as 6-fluoropyridoxal (6-FPAL), 6-fluoropyridoxal phosphate (6-FPLP), and 6-fluoropyridoxine, showed positive heteronuclear 1H-19F nuclear Overhauser effects between the 5'-protons and the 6-fluorine. Apophosphorylase reconstituted with 6-FDPL showed 1% of the activity of the native enzyme in the presence of phosphite. The kinetic pattern, apparent pH optimum of activity, and the activity-temperature dependency of the 6-FDPL-enzyme were virtually identical with those of phosphorylase reconstituted with the parent compound, 6-FPAL [Chang, Y. C., & Graves, D. J. (1985) J. Biol. Chem. 260, 2709-2714], except the Km of phosphite toward the 6-FDPL-enzyme was 9 times higher than that with the 6-FPAL-enzyme and the 6-FDPL-enzyme showed a lower Vmax value. Phosphorylase reconstituted with 5'-deoxypyridoxal (DPL) also showed activity in the presence of phosphite. The kinetics and the temperature-activity dependency of this reconstituted enzyme were investigated. 19F nuclear magnetic resonance studies showed that the binding of glucose 1-phosphate to a 6-FDPL-enzyme-adenosine 5'-phosphate (AMP) complex shifted the 19F signal 0.6 ppm upfield, whereas a 2.1 ppm change was observed when the 6-FPAL-enzyme-AMP formed a complex with glucose 1-phosphate [Chang, Y. C., Scott, R. D., & Graves, D. J. (1986) Biochemistry 25, 1932-1939].(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

The chemical and spectroscopic properties of 6-fluoropyridoxal 5'-phosphate, of its Schiff base with valine, and of 6-fluoropyridoxamine 5'-phosphate have been investigated. The modified coenzymes have also been combined with the apo form of cytosolic aspartate aminotransferase, and the properties of the resulting enzymes and of their complexes with substrates and inhibitors have been recorded. Although the presence of the 6-fluoro substituent reduces the basicity of the ring nitrogen over 10 000-fold, the modified coenzymes bind predominately in their dipolar ionic ring forms as do the natural coenzymes. Enzyme containing the modified coenzymes binds substrates and dicarboxylate inhibitors normally and has about 42% of the catalytic activity of the native enzyme. The fluorine nucleus provides a convenient NMR probe that is sensitive to changes in the state of protonation of both the ring nitrogen and the imine or the -OH group of free enzyme and of complexes with substrates or inhibitors. The NMR measurements show that the ring nitrogen of bound 6-fluoropyridoxamine phosphate is protonated at pH 7 or below but becomes deprotonated at high pH around a pKa of 8.2. The bound 6-fluoropyridoxal phosphate, which exists as a Schiff base with a dipolar ionic ring at high pH, becomes protonated with a pKa of approximately 7.1, corresponding to the pKa of approximately 6.4 in the native enzyme. Below this pKa a single 19F resonance is seen, but there are two light absorption bands corresponding to ketoenamine and enolimine tautomers of the Schiff base. The tautomeric ratio is altered markedly upon binding of dicarboxylate inhibitors. From the chemical shift values, we conclude that during the rapid tautomerization a proton is synchronously moved from the ring nitrogen (in the ketoenamine) onto the aspartate-222 carboxylate (in the enolimine). The possible implications for catalysis are discussed.

19F NMR spectroscopic properties of glycogen phosphorylase reconstituted with 6-fluoropyridoxal (6-FPAL) and 6-fluoropyridoxal phosphate (6-FPLP) were investigated. Analysis of the contribution of chemical shift anisotropy to the line width of the 6-FPLP-enzyme signal shows that the coenzyme molecule is tightly bound to the protein. The chemical shift of the fluorine nucleus in the free 6-FPLP protein is pH independent from pH 6 to pH 9.1. When the 6-FPLP-enzyme forms complexes with AMP, AMP plus glucose-1-P, and AMP plus inorganic phosphate, signals at -11.0, -13.1, and -10.4 ppm are observed, respectively. These different chemical shifts indicate that the protein in each complex has a distinct conformation. The exchange rate between the 6-FPLP-protein-AMP complex and the same complex with bound glucose-1-P is estimated to be 3300 +/- 700 s-1, and that between the 6-FPLP-protein-AMP complex and with bound inorganic phosphate is 500 +/- 100 s-1. The former exchange rate is 13 times faster than that of the same process for the 6-FPAL-enzyme. Analysis of the effects of temperature on the 19F line shape of the 6-FPLP enzyme in the presence of ligands shows that the exchange rates between different complexes drop significantly between 20 and 10 degrees C. Within this temperature range, Arrhenius plots of the enzymatic activities of the native and 6-FPLP-enzymes at varied temperatures also show a pronounced curvature.(ABSTRACT TRUNCATED AT 250 WORDS)