A sensitive automated procedure for the determination of glucuronic acid.

A newly automated method for the detection of uronic acids, particularly glucuronic acid is described. Data showing the limitations of this method are also presented. The advantages of the method are: (1) greatly increased sensitivity compared to the carbazole methods and increased range; (2) lowered interference from other sugars, proteins and salts; (3) variability to allow small sample volumes and/or continuous flow; (4) a more stable color reagent solution.

Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta.

The properties of cyclic nucleotide phosphodiesterase were studied in soluble and particulate fractions from the central nervous system of Manduca sexta (Lepidoptera: Sphingidae). It was determined that: (1) The highest levels of phosphodiesterase occur in nervous tissue. (2) The total and specific enzyme activities of larval and adult brains are greater than those of the remaining ganglia. (3) Specific central nervous sy stem phosphodiesterase activities of the adult are lower than those of the larva, but both protein and total phosphodiesterase contents are considerably greater in the adult central nervous system. (4) Mg2+ is not absolutely required for either cyclic AMP-phosphodiesterase or cyclic GMP-phosphodiesterase activity. (5) Phosphodiesterase is inhibited by a variety of physiological and non-physiological compounds, nucleoside triphosphates being particularly effective; Some potent inhibitors of mammalian phosphodiesterase are comparatively ineffective toward Manduca sexta phosphodiesterase. (6) Kinetic analyses of soluble and particulate phosphodiesterase revealed non-linear double-reciprocal plots for the hydrolysis of both cyclic AMP and cyclic GMP, with Michaelis constants of approximately 10 mu M and 20 mu M; (7) The hydrolysis of both cyclic nucleotides appears in part to be the function of a single enzyme or related enzymes in the insect central nervous system. It follows that the intracellular level of one cyclic nucleotide may influence the concentration of the other by inhibiting its DEGRADATION.

Cyclic nucleotide-stimulable protein kinases in the central nervous sytem of Manduca sexta.

Cyclic nucleotide-stimulable protein kinase (EC 1.7.1.37) has been studied in crude extracts from the central nervous system of the tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae). The insect kinase was fulfhydryl-sensitive and required Mg-2+ for optimal activity. Polyacrylamide gel electrophoresis of supernatants demonstrated the presence of multiple kinases in the larval nerve cord. At low concentrations, cyclic AMP was a much more potent activator of soluble and particulate activities than was cyclic GMP. The specific activity of coluble kinase and the magnitude of its activations by cyclic AMP were greater in the adult than in the larval central nervous system. The exogenous protein substrate specificity of the insect enzyme was similar to that of rat brain kinase with the sole exception that protamine was more readily phosphorylated than histone by nerve cord kinase. It was observed that cyclic AMP lowered the Km of Manduca sexta kinase for ATP, a phenomenon which is apparently nervous tissue=specific in mammals. An effective inhibitor of cyclic AMP-dependent protein kinase was prepared from the larval central nervous system.

Phosphoprotein phosphatase in the central nervous system of Manduca sexta.

The existence and some enzymological properties of phosphoprotein phosphatase (EC 3.1.3.16) have been established in the larval central nervous system of the tobacco hornworm, Manduca sexta (Lepidoptera: Sphingidae). A simple, sensitive and reproducible assay employing 32-P-labeled protamine as a phosphoprotein substrate was employed to measure phosphatase activity in both soluble and particulate fractions of the insect nerve cord. The specific activity of soluble phosphatase in the Manduca sexta central nervous system is of the same order of magnitude as that in mammalian brain. Nerve cord phosphoprotamine phosphatase activity may be stimulated by a variety of monovalent salts, the optimal concentration of NaCl or KCl being 0.2 molar. Activity does not appear to be dependent on bivalent metals and is stimulated by EDTA. A reduced sulfhydryl group is obligatory for maximum activity. Phosphatase could be greatly inhibited by sodium fluoride, ATP and GTP. Cyclic AMP and cyclic GMP are without effect on enzyme activity. Although most of the phosphatase activity in the insect nerve cord appears to be of cytosolic origin, much latent activity can be unmasked by incubating membranous fractions with Triton X-100. In contrast to soluble phosphatase, the detergent-solubilized activity is moderately stimulated by Mn-2+.?