Sachiko Okuno

Growth phase‐dependent changes in the subcellular localization of pre‐B‐cell colony‐enhancing factor1

A cDNA encoding the homolog of the human pre‐B‐cell colony‐enhancing factor (PBEF), a cytokine‐like secreted protein, was isolated from a rat cDNA library. This protein existed in both the cytoplasm and nucleus of the cells, and the amount was higher in the cytoplasm than in the nucleus of proliferating PC‐12 and Swiss 3T3 cells but higher in the nucleus than in the cytoplasm of the PC‐12 cells treated with nerve growth factor and the 3T3 cells grown to a confluent state. Thus, the so‐called PBEF is not a cytokine‐like secreted protein but an intracellular protein associated with the cell cycle.

An immunohistochemical study of Ca2+/calmodulin-dependent protein kinase IV in the rat central nervous system : light and electron microscopic observations

We observed the distribution pattern of Ca2+/calmodulin-dependent protein kinase IV in rat brain and spinal cord using an immunohistochemical method by light and electron microscopy. Particularly strong immunoreactivity was detected in the telencephalic structures such as the olfactory bulb, cerebral cortex, hippocampal formation, caudate-putamen, most nuclei of the dorsal thalamus and the granule cell layer of the cerebellum. Relatively weak staining was observed in the amygdaloid body, some neuron groups of the brainstem reticular formation, the inferior olivary nucleus and the posterior horn of the spinal cord. Immunohistochemical reactivity was not detected in the globus pallidus, substantia nigra, sensory and motor nuclei of the cranial nerves, or in the spinal cord anterior horn. Overall, the distribution of Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity broadly paralleled the sites of expression of signals for messenger RNA of this enzyme. At the subcellular level, Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity appeared exclusively in the nuclei of neurons in the various brain regions, and immunopositive reactivity, although less strong, was also observed in dendritic processes, as well as on the granular endoplasmic reticulum in neuronal somata in these areas. Axon terminals, however, did not show immunoreactivity. These studies demonstrate that Ca2+/calmodulin-dependent protein kinase IV-like immunoreactivity is distributed widely in the central nervous system. The significance of the localization of this enzyme in nuclei is discussed in relation to gene expression.

Maize leaf phosphoenolpyruvate carboxylase: phosphorylation of Ser15 with a mammalian cyclic AMP-dependent protein kinase diminishes sensitivity to inhibition by malate

The so‐called light‐activation of phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) involved in C4 photosynthesis is known to be mediated by phosphorylation. A cyclic AMP‐dependent protein kinase from bovine heart was found to be able to phosphorylate PEPC. The phosphorylation was accompanied by the changes in kinetic properties, which were very similar to the reported light activation. The phosphorylated amino acid residue was identified as Ser and the position of this Ser on the primary structure [(1988) FEBS Lett. 229, 107‐110] was determined to be Ser15.

Critical amino acid residues of AIP, a highly specific inhibitory peptide of calmodulin-dependent protein kinase II

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin‐dependent protein kinase II (CaMKII), was studied. Replacement of Arg6, Gln7, or Ala9 by other amino acid residues produced a marked increase in the IC50 value. Leu4 and Val10 were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala3, Glu8, Ala12, and Leu13 by other residues produced no significant increase in the IC50, the substitution of Lys for Ala3 decreased the IC50. An AIP analog (KK LRRQEA DAY), in which Ala3 and Val10 were replaced with Lys and Phe, respectively, showed an IC50 value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.

Assay of tyrosine 3-monooxygenase using the coupled nonenzymatic decarboxylation of dopa

Dopa was found to be decarboxylated nonenzymatically by ferricyanide over the range of pH 4.5 to 10. On the basis of this finding, a new assay method of tyrosine 3-monooxygenase using the coupled nonenzymatic decarboxylation by ferricyanide was developed. The new method was excellent not only in sensitivity and reliability, but also in convenience.

Localization of the mRNAs for two isoforms of Ca2+/calmodulin-dependent protein kinase kinases in the adult rat brain.

Ca2+/calmodulin-dependent protein kinase (CaM kinase) I and IV are thought to be activated by CaM kinase kinases (CaMKK). We examined the distribution of mRNAs for two isoforms (alpha and beta) of CaMKKs in the brain by in situ hybridization histochemistry. In the adult rat brain, CaMKK alpha mRNAs are widely distributed throughout the brain, whereas CaMKK beta mRNAs are restricted to some neuronal populations, particularly the cerebellar granule cells.

Accurate assay of dopa decarboxylase by preventing nonenzymatic decarboxylation of dopa

The nonenzymatic decarboxylation of dopa was completely blocked by both 2-mercaptoethanol and EDTA together over the wide range of pH. This finding made it possible to measure the activity of dopa decarboxylase precisely even at an alkaline pH value. The pH optimum of dopa decarboxylase was found to be pH 7.0 and the Km value for dopa was determined to be 4 X 10(-5) M.

STABILIZATION, PURIFICATION AND CRYSTALLIZATION OF CATALYTIC SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE FROM BOVINE HEART

The catalytic subunit of cAMP-dependent protein kinase purified from bovine heart was significantly stabilized by non-ionic detergents. Freezing and thawing did not cause a significant decrease in the enzyme activity in the presence of 0.1% Tween 80. A very convenient method for purification and crystallization of the catalytic subunit was also described.

Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase

The tryptophan hydroxylase activity of the crude extract from rat brain stem was stimulated approximately 2‐fold by incubation with cAMP analogues under protein phosphorylating conditions. The cAMP‐dependent activation process of the enzyme needed not only cAMP‐dependent protein kinase but also activator protein. The kinetic properties of the enzyme activated by cAMP‐dependent protein kinase were very similar to those of the enzyme activated by calmodulin‐dependent protein kinase II.

Distribution of Ca2+/calmodulin-dependent protein kinase α in the rat central nervous system: an immunohistochemical study

Abstract Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated by Cam-kinase IV kinase. We provided a rabbit antiserum against 20 amino acid residues at the carboxyl-terminal end of CaM-kinase IV kinase, and examined regional and intracellular distribution of CaM-kinase IV kinase immunohistochemically in the central nervous system of the rat by light and electron microscopy. The immunoreactivity was found in cellular nuclei of virtually all neurons. However, the immunoreactivity was weak in the nuclei of the granule cells in the cerebellar cortex, although the nuclei of the granule cells were reported to contain high CaM-kinase IV activity. Thus, it was suggested that other types of CaM-kinase IV kinase might exist in the cerebellum, and the present CaM-kinase IV kinase was named as CaM-kinase kinase α.