An improved procedure of the solubilization and purification of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) from bovine cerebral white matter is reported. To remove easily extractable protein, the tissue was homogenised in 10 vol. of 0.5 M‐ammonium acetate containing 10 mM‐Tris. HCI, pH 6.9, at 4°C and centrifuged at 105,000 g for 60 min. The precipitate was extracted with 10 vol. of 0.5% Triton X‐100 containing 10 mM‐Tris. HCI, pH 6.9, and centrifuged, By this extraction, over 70% soluble protein could be removed in the supernatant and most CNPase activity was kept in the precipitate. The precipitate was extracted with 10 vol. of 1% Triton X‐100 and 1 M‐ammonium acetate mixture containing 10 mM‐Tris.HCI, pH 8.2, and centrifuged at 105,000 g for 60 min. The extract contained 54% of CNPase and the specific activity was fivefold that of the original homogenate. Subsequently, the extractions were carried out with 2% Triton X‐100‐2 M‐ammonium acetate and 4% Triton X‐100‐4 M‐ammonium acetate at pH 8.2. The recovery of CNPase was found to be nearly 90% from the original homogenate, without loss of enzyme activity during extraction, while much CNPase activity was lost when guanidinium chloride was used as the extraction medium. Using the Triton X‐100‐ammonium acetate extract, several column chromatography techniques were applied to purify the enzyme. In the first step, Phenyl‐Sepharose CL‐4B column chromatography was performed by eluting with a double‐linear gradient of ammonium acetate and Triton X‐100. In the second step, the fraction containing CNPase after Phenyl‐Sepharose CL‐4B column chromatography was applied to a Sepharose 6B column and the enzyme was eluted with 1% Triton X‐100‐ I M‐ammonium acetate, pH 8.2. The peak containing CNPase was applied to CM‐Sepharose CL‐6B column chromatography in the final step. The enzyme was eluted with a linear gradient of KCI. In this step, CNPase eluted as a sharp peak and the specific activity was approximately 2300 pmol 2′‐AMP formed/min/mg protein. The recovery of CNPase from the original homogenate was about 50%. By the isoelectrofocusing technique, the pI of CNPase was found to be 8.6. When Reisfeld polyacrylamide gel electrophoresis and SDS‐polyacrylamide gel electrophoresis were carried out on the purified CNPase, only one protein band, corresponding to CNPase activity, was detected. Its molecular weight was estimated to be approximately 51,000 as the active enzyme form. K, value of the purified enzyme for 2′,3′‐CAMP calculated from a Lineweaver‐Burk plot was 3.13 mM.

We examined the influence of electroconvulsive seizure (ECS) and imipramine (IMI) treatment on the transcription and translation of cyclic nucleotide phosphodiesterase type IV (PDE IV) isozymes in the rat brain. Our in situ hybridization studies revealed an increase of PDE IV-B mRNA level in various brain regions after acute ECS. However, the increase of PDE IV activity was produced not by acute but by chronic ECS treatment in the frontal cortex. Increased PDE V-B mRNA expression in frontal but not in hippocampal subfields was induced also after chronic ECS treatment. Although an increase in PDE V-A mRNA expression of the dentate gyrus in the hippocampus was observed, no change of PDE IV activity was produced in the hippocampus by acute or chronic ECS treatment. These results suggest that the repeated increases of PDE lV-B mRNA expression are attributable to the increase of PDE IV translation. Increased PDE IV-B transcription and PDE IV translation in the frontal cortex were also produced after chronic Ml treatment. This is the first report demonstrating an expressional regulation of Drosophila melanogaster dunce (dnc) gene homologue PDE IV isozymes in the brain. Although no pathophysiological conditions with reduced PDE IV activity in the nervous system are known except for a learning deficit in the mutant fly dnc -, our results suggest possible treatments to cope with reduced PDE IV activity. Key Words: Cyclic nucleotide phosphodiesterase type IV-Rolipram -Electroconvulsive seizure-Imipramine-In situ hybridization-Enzyme activity.

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We have succeeded in operating a transition-edge sensor (TES) spectrometer and evaluating its performance at the SPring-8 synchrotron x-ray light source. The TES spectrometer consists of a 240 pixel National Institute of Standards and Technology (NIST) TES system, and 220 pixels are operated simultaneously with an energy resolution of 4 eV at 6 keV at a rate of ∼1 c/s pixel−1. The tolerance for high count rates is evaluated in terms of energy resolution and live time fraction, leading to an empirical compromise of ∼2 × 103 c/s (all pixels) with an energy resolution of 5 eV at 6 keV. By utilizing the TES’s wideband spectroscopic capability, simultaneous multi-element analysis is demonstrated for a standard sample. We conducted x-ray absorption near-edge structure (XANES) analysis in fluorescence mode using the TES spectrometer. The excellent energy resolution of the TES enabled us to detect weak fluorescence lines from dilute samples and trace elements that have previously been difficult to resolve due to the nearly overlapping emission lines of other dominant elements. The neighboring lines of As Kα and Pb Lα2 of the standard sample were clearly resolved, and the XANES of Pb Lα2 was obtained. Moreover, the x-ray spectrum from the small amount of Fe in aerosols was distinguished from the spectrum of a blank target, which helps us to understand the targets and the environment. These results are the first important step for the application of high resolution TES-based spectroscopy at hard x-ray synchrotron facilities.