[Ketone bodies: measurement and its clinical significance].
Explore the source record for details and available documents.
Humans↗
Biomedical subjects
H Hidaka
Publications and source records attributed to H Hidaka.
Activation mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-kinase IV kinase.
This manuscript examines the mechanisms by which Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) is activated through the binding of Ca2+/CaM and by phosphorylation. Studies with the synthetic autoinhibitory domain peptides of CaM-kinase II indicate that CaM-kinase IV has a similarly located autoinhibitory domain, and this was confirmed since site-directed mutagenesis of this region (HMDT308 to DEDD and FN317 to DD) generated fully active Ca2+/CaM-independent kinases. Total activities of purified, baculovirus-expressed wild type and mutant kinases were increased 2-fold by intramolecular autophosphorylation, but this reaction was extremely slow (1-2 h) and probably not physiological. However, CaM-kinase IV can be activated by brain CaM-kinase IV kinase resulting in large increases in both total (5-7-fold) and Ca2+/CaM-independent (> 20-fold) CaM-kinase IV activities. This activation reaction required Mg2+/ATP and Ca2+/CaM, was intermolecularly catalyzed, and was reversed by protein phosphatase 2A. Activation of CaM-kinase IV resulted in a 10-fold decrease in Km for syntide-2 with little effect on Km for ATP or Vmax. CaM-kinase IV kinase was highly purified from rat brain extract and was shown to be a 68-kDa monomer. The results of this study demonstrate that CaM-kinase IV does have an autoinhibitory domain within residues His305-Lys321 that suppresses kinase activity in the absence of Ca2+/CaM. CaM-kinase IV is not significantly activated by autophosphorylation, but it can be activated 10-fold by a CaM-kinase IV kinase. This kinase cascade activation mechanism may be important for the physiological function of CaM-kinase IV such as transcriptional regulation through phosphorylation of cAMP responsive element binding protein (Enslen, H., Sun, P., Brickey, D., Soderling, S. H., Klamo, E., and Soderling, T.R. (1994) J. Biol. Chem. 269, 15520-15527).
Amino Acid Sequence↗
Differential distribution of neurocalcin isoforms in rat spinal cord, dorsal root ganglia and muscle spindle.
The cellular distribution of neurocalcin isoforms in rat spinal cord, dorsal root ganglia and muscle spindle was examined using immunohistochemical techniques and two antibodies against different isoforms. In the study using the antibody against the purified neurocalcin from bovine brain, nerve fiber terminals and the cytoplasm of small-sized neurons in the posterior horn, some medium-sized neurons and numerous axons were strongly stained. In dorsal root ganglia, the cytoplasm of various-sized neurons, including about 40% of the total neurons, was also stained. Sensory nerve endings in muscle spindle were immunopositive. When the antibody against the expressed neurocalcin delta was applied, nuclei of the neurons were mainly stained in the spinal cord and dorsal root ganglion. The cytosol of glial cells but not axons in white matter showed neurocalcin delta-like immunoreactivity against the antibody. These findings suggest that each of the differently located isoforms may play a distinct role in Ca2+ signaling in sensory systems.
Amino Acid Sequence↗
Biphasic effect of estrogen on neuronal constitutive nitric oxide synthase via Ca(2+)-calmodulin dependent mechanism.
Estrogen is known to retard the development of atherosclerosis and to work in the brain, but the mechanism of hormonal action is completely unknown. We investigated the effect of estrogen on the activity of neuronal constitutive nitric oxide synthase (NNOS). A low concentration of estrogen (10(-10)(-7) M) enhanced the activity of homogenates of the cytosol fraction of rabbit cerebellums and also that of partially purified NNOS, and high dose (10(-6)(-5) M) attenuated them. The study using estrogen receptor antagonists, tamoxifen, clomiphene, and ICI182780 suggested that estrogen receptor did not relate significantly to those effects of 17 beta-estradiol. 17 alpha-estradiol or progesterone did not change significantly it in low doses, although moderately inhibited it in high doses. Estrogen enhanced the fluorescence of dansyl-calmodulin in low doses and attenuated it in high doses, suggesting that estrogen affects Ca(2+)-calmodulin directly. This study demonstrated that estrogen has a biphasic effect on the activity of NNOS through a Ca(2+)-calmodulin.
Amino Acid Oxidoreductases↗
Ca2+/calmodulin-dependent protein kinase kinase cascade.
Recently we obtained evidence for the existence of Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), which was partially purified in the process of the purification step of Ca2+/calmodulin-dependent protein kinase V (CaM kinase V). This enzyme promoted phosphorylation of the autophosphorylation site on CaM kinase V and activated the activity (Mochizuki, H. et al. (1993) Biochem. Biophys. Res. Commun. 197, 1595-1600). Present study revealed that CaMKK also phosphorylated CaM kinase IV associated with the activation of its activity. Phosphorylation of CaM kinase IV by CaMKK occurred on multiple sites. Furthermore, CaM kinase IV and CaM kinase V phosphorylated each other which resulted in their activation. The phosphorylation site of CaM kinase V by CaM kinase IV was the same as the autophosphorylation site. Our study suggests the existence of a heretofore CaM kinases cascade consisting of a sequential activation of each CaM kinase.
Adenosine Triphosphate↗
Development-related and cell-type specific nuclear localization of annexin XI: immunolocalization analysis in rat tissues.
Subcellular distribution of a novel annexin, annexin XI, was examined in rat tissues. As we have previously reported, annexin XI mainly localizes in nuclei of 3Y1, the rat embryonic fibroblast cell line; however, immunoperoxidase staining was not particularly nuclear in most of the tissues examined in adult rats. Nuclear localization of annexin XI was uncommon in adult rat tissues. By contrast, in the day-14 rat embryo, the undifferentiated mesenchymal cells were labeled mainly in the nuclei. The connective tissues of 18-day fetus, however, did not show predominantly nuclear staining any longer. In addition, the developing gray matter of the embryonic rat spinal cord exhibited primarily nuclear localization of annexin XI while the annexin XI immunoreactivity diminished and became absent from the nuclei in the adult spinal cord. On the other hand, the endodermal cells never displayed nuclear annexin XI at any developmental stages examined. All these findings suggested that subcellular localization of annexin XI is regulated depending on developmental stages and the cell types.
Animals↗
Ca2+/calmodulin-dependent protein kinase V and I may form a family of isoforms.
We reported that polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) reacted to two proteins of rat cerebrum with a molecular mass of 40 and 41 kDa. This antibody revealed the immunoreactivity with CaM kinase I expressed in E. coli (recombinant CaM kinase I), of which molecular mass was 40 kDa, whereas 41 kDa mainly with purified CaM kinase V. The immunoreactive bands of recombinant CaM kinase I and CaM kinase V did not shift by phosphorylation or dephosphorylation. These results suggest that CaM kinase V and CaM kinase I may form a family of isoforms.
Animals↗
The effect of KN-62, Ca2+/calmodulin dependent protein kinase II inhibitor on cell cycle.
The isoquinolinesulfonamide derivative, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin dependent protein kinase II (CaM kinase II) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M., and Hidaka, H.(1990) J. Biol. Chem. 265, 4315-4320). KN-62 inhibits growth of K562 cells, in a dose-dependent manner. Flow cytometric analysis demonstrates that the treatment of K562 cells with 10 microM KN-62 causes an accumulation of cells in S phase. Immunoblotting studies showed that specific antibodies against CaM kinase II recognized the 65 kDa of protein in K562 cells. This protein showed protein kinase activity as examined by the activity gel method. The inhibition of this enzyme activity by KN-62 was dose-dependent. The immunoprecipitates with the antibodies from K562 cells phosphorylates the synthetic peptide substrates, syntide-2. These results suggest that CaM kinase II plays an important role in the mechanisms for the cell growth in K562 cells.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
A role of calcyclin, a Ca(2+)-binding protein, on the Ca(2+)-dependent insulin release from the pancreatic beta cell.
Exocytosis is an important example of cell activation. Ca2+ and calcium-binding proteins are considered to modulate signal transduction in exocytosis. We examined the role of calcyclin, calcium-binding protein, in the stimulus-secretion coupling in pancreatic beta cells. The presence of calcyclin in these cells was demonstrated immunologically. We permeabilized rat pancreatic islets with streptolysin-O and examined the effects of calcyclin on insulin release. The Ca(2+)-stimulated insulin release was enhanced by calcyclin, in a dose-dependent manner, whereas this calcium-binding protein had no effect on insulin release from islets in low Ca2+ buffer or from the islets not subjected to the streptolysin-O treatment. Calgizzarin, another member of the S-100 protein family, had no apparent effect on the Ca(2+)-stimulated secretion under parallel conditions. An anticalcyclin antibody suppressed the increase in insulin release induced by calcyclin. We propose that calcyclin may be involved in signal transduction of the Ca(2+)-induced release of insulin.
Animals↗
Isolation and characterization of a 36-kDa microfibril-associated glycoprotein by the newly synthesized isoquinolinesulfonamide affinity chromatography.
We have reported here the biochemical characterization of a newly identified microfibril-associated protein of 36-kDa (36-kDa MAP) from bovine aorta. Using Ca(2+)-dependent affinity chromatography on an isoquinolinesulfonamide derivative (CKA-1303)-coupled Sepharose, we obtained a pure form of 36-kDa MAP. This compound should serve as a useful tool for clarifying the physiological roles of 36-kDa MAP. In addition, 45Ca-autoradiography clearly indicated that 36-kDa MAP binds Ca2+. Partial amino acid sequence of 36-kDa MAP was determined. Our search of that published sequence against a 36-kDa MAP sequence revealed the resemblance to fibrinogen-related proteins of sea cucumber, cytotactin and tenascin which are substrate-adhesion proteins made at critical stages of histogenesis.
Amino Acid Sequence↗
Purification, characterization, and partial sequence analysis of a new 25-kDa actin-binding protein from bovine aorta: a SM22 homolog.
We have purified a Ca(2+)-sensitive 25-kDa protein from bovine aorta. The 25-kDa protein remains associated with the membrane fraction in the presence of Ca2+ and is dissociated by EGTA. The purified protein binds directly to F-actin at a ratio of 1:6 actin monomers, with a binding constant of 7.0 x 10(5) M-1. The partial sequence analysis revealed a high homology to predicted protein derived from mRNA, named WS3-10 and chicken SM22 protein. Although chicken SM22 had not any interaction with contractile proteins or Ca2+, the bovine homolog clearly binds to F-actin and has Ca(2+)-binding domain in its primary structure.
Actins↗
Induction of phagocytic activity of M1 cells by an inhibitor of vacuolar H+-ATPase, bafilomycin A1.
Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, time- and dose-dependently induced the differentiation of M1 cells, a murine myeloid leukemic cell line, into macrophage-like cells as revealed by the phagocytosis of polystyrene latex particles. This differentiation was inhibited not only by actinomycin D and cycloheximide but also by ST-638 (an inhibitor of tyrosine kinase). However, it was affected neither by K-252a (an inhibitor of C-kinase) nor by H-89 (an inhibitor of A-kinase), in contrast to the M1 cell differentiation induced by leukemia inhibitory factor (LIF). Okadaic acid inhibited both the bafilomycin A1-induced and LIF-induced differentiation of M1 cells.
Animals↗
Characterization of gamma- and delta-subunits of Ca2+/calmodulin-dependent protein kinase II in rat gastric mucosal cell populations.
We searched for the occurrence of a Ca2+/calmodulin-dependent protein kinase in rat gastric cell types as a likely member in the chain of gastrin- and muscarinic-receptor-mediated signal transmission. A Ca(2+)- and calmodulin-dependent phosphorylation of major 50, 60 and 100 kDa substrates was observed in parietal cell cytosol and a major 60 and 61 kDa protein doublet was found to bind 125I-calmodulin in 125I-calmodulin-gel overlays. A specific substrate of the multifunctional Ca2+/calmodulin-dependent protein kinase II, autocamtide II, was phosphorylated in a calmodulin-dependent manner. The specific inhibitor of this enzyme, KN-62, antagonized protein kinase activity. RNA extracted from gastric mucosal cells was shown to contain sequences of the gamma- and delta- but not alpha- and beta-subunits of the calmodulin-dependent kinase II, and mRNA of both subtypes was demonstrated in highly purified parietal, chief and mucous cells. A calmodulin-dependent kinase II composed of gamma- and delta-subunits is a likely mediator of Ca(2+)-dependent signal transmission in these populations of gastric cells.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Evidence for posttranscriptional regulation of transgenic protein kinase C-alpha in T cells.
Recently, we succeeded in establishing a transgenic mouse line which expressed high levels of protein kinase C (PKC)-alpha in thymocytes at the mRNA level with disproportionately small increases at the protein level. The transgenic PKC-alpha was nevertheless functionally active for inducing accelerated cell growth and IL-2 production by stimulation with anti-receptor (CD3) antibody or phorbol 12-myristate 14-acetate (PMA) in vitro. Study of the dynamics of transgenic PKC-alpha in the cells in vitro showed that the amount of PKC-alpha protein increased in the cells remarkably at > or = 5 h after stimulation, whereas the level of PKC-alpha mRNA did not change significantly or changed slightly. This suggested that cell activation breaks the posttranscriptional regulation of the transgenic PKC-alpha in resting cells. The increase in PKC-alpha protein accompanied a prolonged membrane translocation of PKC-alpha and enhanced cell proliferation. Such a transgenic effect was inhibited completely by a PKC inhibitor, H-7, added during 0-6 h after the stimulation. These results show formally that the transgenic PKC-alpha whose production was accelerated through cell activation plays a key role in the late (for > or = 5 h) signal delivery for disregulated cell growth.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Kinase requirement for retinal growth cone motility.
Since cytoplasmic Ca2+ levels are reported to regulate neurite elongation, we tested whether calcium-activated kinases might be necessary for growth cone motility and neurite elongation in explant cultures of goldfish retina. Kinase inhibitors and activators were locally applied by micropipette to retinal growth cones and the responses were observed via phase-contrast videomicroscopy. In some cases, growth rates were also quantified over several hours after general application in the medium. The selective inhibitors of protein kinase C, calphostin C (0.1-1 microM) and chelerythrin (up to 50 microM), caused no obvious changes in growth cones or neurite elongation, and activators of PKC (phorbols, arachidonic acid, and diacylglycerol) also were generally without effects, although phorbols slowed the growth rate. Inhibitors of protein kinase A and tyrosine kinases also produced no obvious effects. The calmodulin antagonists, calmidazolium (0.1 microM), trifluoperazine (100 microM), and CGS9343B (50 microM), however, caused a reversible growth cone arrest with loss of filopodia and lamellipodia. The growth cone became a club-shaped swelling which sometimes moved a short distance back the shaft, leaving evacuated filaments at points of strong filopodial attachments. A similar reversible growth cone arrest occurred with the general kinase inhibitors: H7 at 200 but not at 100 microM, and staurosporine at 100 but not 10 nM, suggesting possible involvement of a calmodulin-dependent kinase (camK) rather than PKC. The selective inhibitor of camKII, KN-62 (tested up to 50 microM), produced no effects, but the specific myosin light-chain kinase (MLCK) inhibitors ML-7 (3-5 microM) and ML-9 (5-10 microM) reversibly reproduced the effect, suggesting that MLCK rather than camKII is necessary for growth cone motility. The MLCK inhibitors' effects both on growth cone morphology and on F-actin filaments (rhodamine-phalloidin staining) were similar to those caused by cytochalasin D (5 microM), and are discussed in light of findings that inhibiting MLCK disrupts actin filaments in astrocytes and fibroblasts.
Actins↗
Ca2+/calmodulin-dependent protein kinase V: tissue distribution and immunohistochemical localization in rat brain.
Polyclonal antibody against Ca2+/calmodulin-dependent protein kinase V (CaM kinase V) was prepared from guinea pigs immunized with synthetic polypeptide, based on the partial amino acid sequence of the rat brain enzyme. Immunoblot analysis of purified CaM kinase V revealed two immunoreactive bands with a molecular mass of 41 kDa and minor 40 kDa, respectively. Tissue distribution of CaM kinase V and immunohistochemical localization in rat brain were also investigated. Immunoblotting revealed the presence of immunoreactive proteins with the same molecular mass of 40 and 41 kDa, in the cerebrum, cerebellum, brain stem, pituitary gland, lung, adrenal gland, spleen, liver, colon, heart, stomach, ovary, spinal cord, and thymus. Immunohistochemistry revealed strong staining in the neuronal somata and weak staining in the nuclei. Densely stained regions included the cerebral cortex, the hippocampal formation, the caudatoputamen, the globus pallidus, the hypothalamus, the substantia nigra, the medial geniculate body, the olfactory bulb, the cerebellar cortex and the choroid plexus. CaM kinase V revealed different substrate specificity compared with CaM kinase II. These results lead to the notion that CaM kinase V may exist in 40- and 41-kDa isoforms, is widely distributed in various tissues, and may play an important role in the control of a wide variety of calcium regulated processes in the respective tissues.
Amino Acid Sequence↗
Ca(2+)-calmodulin mediated modulation of the electrical coupling of ventricular myocytes isolated from guinea pig heart.
The mechanism for the regulation of junctional conductance (gj) by Ca2+ was examined in paired ventricular myocytes isolated from guinea pig heart. One cell of the pair was voltage-clamped by a single-patch pipette, and gj was measured after perforation of the non-junctional membrane of the partner cell. The average value of gj under control condition at pCa 9.0 was 181 +/- 13 nS (n = 30). An elevation of calcium concentration ([Ca2+]i) in the intracellular perfusate from pCa 9.0 to pCa 5.7 resulted in a decrease of 32 +/- 5% in gj (n = 18). In myocytes pretreated with W7 (10(-4) M), a similar elevation of [Ca2+]i caused a decrease of only 10 +/- 3% in gj (n = 6), indicating a protective action of W7 against Ca(2+)-mediated electrical uncoupling. W5 (10(-4) M), a non-chlorinated derivative of W7, did not show such a protective action. Calmodulin (10(-5) M) had no effects on gj at pCa 9.0. However, at moderately elevated [Ca2+]i condition at pCa 7.0, calmodulin (10(-5) M) decreased gj by 29 +/- 6% (n = 4). These results suggest that calmodulin may play an important role in the Ca(2+)-mediated regulation of gap junctional channel function in the cardiac ventricular cells.
Action Potentials↗
Immunocytochemical detection of Ca(2+)-dependent subspecies of protein kinase C in mouse embryos before and during compaction.
To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca(2+)-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.
Animals↗