[Phospholipase D in signal transduction].
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Alcoholism↗
Biomedical subjects
H Kanoh
Publications and source records attributed to H Kanoh.
Purification and characterization of cytosolic diacylglycerol kinases of human platelets.
Three isozymes of diacylglycerol kinase (DGK), DGK-I, DGK-II, and DGK-III, were purified from the cytosol of human platelets by successive chromatography on DEAE-cellulose, Ultrogel AcA34, heparin-Sepharose, ATP-agarose, Mono Q, phenyl-Superose, HCA-hydroxyapatite, Wakopak G40, and TSK-3000SW columns. Two DGK species (DGK-I and DGK-III) were purified to apparent homogeneity, and upon SDS-polyacrylamide gel electrophoresis, they showed a single band of apparent molecular mass of 152 kDa (DGK-I) or 58 kDa (DGK-III). The peptide mapping analysis showed that DGK-I and DGK-III are structurally different. DGK-II was only partially purified, and its apparent Mr was estimated to be 75,000 by gel filtration. The specific enzyme activities of the three isozymes were increased 1,480-fold (DGK-I), 690-fold (DGK-II) and 2,100-fold (DGK-III) over original platelet cytosol. The activities of DGK-II and DGK-III were markedly enhanced by the presence of deoxycholate or phosphatidylserine, whereas DGK-I activity was not much affected by the anionic compounds. All of the three activities were strongly suppressed by phosphatidylcholine. Triton X-100 and octyl glucoside were strongly inhibitory to all of the enzymes, although to different extents. The DGK inhibitor, R59022, inhibited DGK-II and to a lesser extent DGK-III, but little affected DGK-I activity. DGK-I was much more heat-stable than DGK-II and DGK-III. The Km values for ATP were 150 microM for DGK-I, 245 microM for DGK-II, and 450 microM for DGK-III. The apparent Km values for suspended diolein were not much different among the DGKs and were in the range of 50-80 microM. These observations indicate that human platelet cytosol contains DGK isozymes with different enzymological properties. Furthermore, the three DGKs isolated from human platelets were found not to cross-react with the antibody raised against porcine brain 80-kDa DGK, thus indicating that human platelets contain novel species of DGK.
Blood Platelets↗
Partial purification and characterization of phosphatidylinositol kinases from human platelets.
Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.
1-Phosphatidylinositol 4-Kinase↗
Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane.
Tetrahymena calmodulin (CaM) differs from mammalian CaM in its ability to activate Tetrahymena guanylate cyclase. Of 12 differences in amino acid sequence, two occur near the carboxyl terminus (Gln-143----Arg and Thr-146----deletion). To investigate the functional significance of the carboxyl-terminal region in activation of the guanylate cyclase, three mutated CaMs were engineered by using cassette mutagenesis of rat CaM cDNA: Gln-143----Arg (CaM.A), Thr-146----deletion (CaM.D), and Gln-143----Arg/Thr-146 deletion (CaM.AD). Recombinant wild type CaM (wCaM), CaM.A, CaM.D, and CaM.AD were indistinguishable in their ability to activate cyclic AMP phosphodiesterase. The two mutated CaMs (CaM.A and CaM.AD) with the Gln-143 replacement activated guanylate cyclase of Tetrahymena plasma membrane in the presence of Ca2+, with the maximal activation being half of that produced by Tetrahymena CaM. In contrast, neither CaM.D nor wCaM could stimulate the cyclase activity. A CaM antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), prevented the cyclase activation by either Tetrahymena CaM, CaM.A, or CaM.AD. Thus, we conclude that Arg-143 is in a region of the molecule involved in activation of Tetrahymena guanylate cyclase. The data also suggest that the cyclase activation by Tetrahymena CaM requires complex macromolecular interactions between the entire CaM molecule and the enzyme.
3',5'-Cyclic-AMP Phosphodiesterases↗
Porcine diacylglycerol kinase sequence has zinc finger and E-F hand motifs.
Cell stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Of the DGK isoforms so far reported, only porcine DGK from lymphocytes has been characterized in detail. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C, and two E-F hand motifs, typical of Ca2(+)-binding proteins like calmodulin. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate.
Amino Acid Sequence↗
Non-heparin-binding endothelial cell growth factor from bovine omentum.
The graft of omental pedicle is known to be clinically effective for wound healing and revascularization of ischemic organs. We found that bovine greater omentum contained growth factor that was capable of stimulating the proliferation of bovine aortic endothelial cells. Gel filtration of the tissue extract showed at least two activity peaks corresponding to molecular weights of 96,000 and 21,000. The major Mr 21,000 growth factor was partially purified approx 120-fold from the omental extract. The purified factor was not mitogenic to BALB/c 3T3 cells and, importantly, had no affinity for immobilized heparin. This factor is thus clearly distinct from fibroblast growth factors and related mitogens. The pI of the factor was estimated to be 5.6-6.0. This factor may be involved in the potent angiogenic activity expressed by the implanted omentum. The omental fat, which was previously shown to cause neovascularization in the assay in vivo, did not promote the growth of endothelial cells in vitro.
Animals↗
Diacylglycerol kinase: a key modulator of signal transduction?
Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.
Animals↗
[Primary culture and characterization of human ovarian surface epithelial cells].
Primary culture and characterization of the human ovarian surface epithelial cells were carried out to make a preliminary trial of the histogenetic consideration of common epithelial tumors of the ovary utilizing in vitro systems. Ovarian surface epithelial cells were harvested from strips of surgically excised ovary using an explant culture method and submitted to studies. The cells appeared flat epithelioid in shape and arranged in a cobblestone pattern. Immunocytochemical stainings by the PAP method revealed that these epithelioid cells were keratin and vimentin positive. The cells grew in the medium of RPMI 1640 with 10% fetal calf serum and the growth rate was accelerated significantly when supplemented with hEGF (10 ng/ml) and hydrocortisone (0.5 microgram/ml). When epithelioid cells were cultivated on Matrigel coated glass, some morphological changes were brought about by treating them with TPA (10 ng/ml) or sodium butyrate (1.5 mM). When the cells were cultivated on type I collagen gel, the arrangement of the cells appeared to be mostly monolayer. Grown in Matrigel overlaid on type I collagen gel, the cells grouped and arranged seemingly in spreading branches. Although no malignant transformation has been observed, some morphological changes reminiscent of metaplastic change were seen in some of the cultured cells when the media were treated repeatedly with 5-azacytidine (3 microM), the DNA hypomethylating agent. The results reported may help to some extent to clarify the biological nature of ovarian surface epithelial cells including ovarian tumorigenesis which still remains unknown.
Adult↗
[Diacylglycerol kinase--a novel enzyme having both zinc finger and E-F hand motifs].
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Adenosine Triphosphate↗
High-level expression of complementary DNA encoding rat calmodulin in Escherichia coli.
We report the production and characterization of a rat calmodulin made in Escherichia coli. To express the rat calmodulin cDNA in E. coli, we have employed an expression vector containing the E. coli trp promoter and trpA terminator. The cDNA was modified so as to delete the 5' nontranslated sequence and to incorporate a consensus sequence for the E. coli ribosome-binding site. Several codons for the N-terminal amino acids were selected to fit the E. coli consensus nucleotide sequence around the translational initiation codon. After induction of expression in E. coli, rat calmodulin accounted for over 30% of total cellular proteins. About 100 mg of recombinant rat calmodulin, purified to over 90% homogeneity by extraction from bacterial lysate followed by phenyl-Sepharose column chromatography, was obtained from 1 liter of E. coli culture. This recombinant calmodulin activated rat brain cyclic AMP phosphodiesterase to the same extent as the native calmodulin purified from rat brain. These results indicate that the overproduction system of the recombinant calmodulin in E. coli facilitates the study of the structure-function relationship by site-specific mutagenesis.
Amino Acid Sequence↗
Different effects of sphingosine, R59022 and anionic amphiphiles on two diacylglycerol kinase isozymes purified from porcine thymus cytosol.
Porcine thymus cytosol contains two immunologically distinct forms of diacylglycerol kinase (DGK) [Yamada, K. and Kanoh, H. (1988) Biochem. J. 255, 601-608]. These 2 DGK species, having apparent molecular masses of 80 and 150 kDa, were purified from the thymus cytosol. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 150-kDa DGK gave 2 polypeptide bands of 50 and 75 kDa, whereas the 80-kDa DGK yielded a single protein band. The 80-kDa DGK was markedly activated by 10-20 microM sphingosine as well as by the known anionic activators such as phosphatidylserine and deoxycholate. In contrast, the 150-kDa DGK was fully active in the absence of the anionic activators and was strongly inhibited by sphingosine (IC50, 20 microM). The putative DGK inhibitor R59022 inhibited the 80-kDa DGK (IC50, 10 microM), but had little effect on the 150-kDa form. It is therefore clear that in the thymus cytosol there are at least 2 DGK isozymes operating under different control mechanisms.
Animals↗
Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.
We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.
Animals↗
Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells.
80 kDa diacylglycerol kinase (DGK) was immunoquantitated in cell homogenates and subcellular fractions. It was extremely abundant in the cytosol of various lymphocytes and comprised, in the highest case, more than 0.2% of the total soluble protein in T cell-enriched pig splenocytes. The lymphocyte membrane contained less than 10% of the total cellular DGK protein. The content of 80 kDa DGK in the human T cell leukemic cell line, Jurkat (360 ng/mg homogenate protein), was similar to those in pig and human peripheral blood lymphocytes. In contrast, the enzyme level was very low in the human promyeloblastic cell line, HL-60 (less than 10 ng/mg homogenate protein), and was undetectable in human polymorphonuclear leukocytes. These findings indicate that the content of 80 kDa DGK is markedly variable depending on the type of cells, even though all these cells are known to accumulate phosphatidate rapidly upon cell stimulation.
Animals↗
Conformational change and aggregation of concanavalin A at high temperatures.
Increase of the beta sheet content and aggregation of concanavalin A (con A) induced at about 60 C were followed with circular dichroism (c.d.) and scattered light intensity (I90) on both metal-complexed and demetallized species. The conversion occurred at a higher temperature for metal-complexed species than for demetallized one. A concentration-independent conversion curve of metal-complexed species, obtained for a concentration range below around 6 microgram/ml (6 x 10(-3) kg m-3) with a midpoint at 57 degrees C, was well described in terms of a conformational equilibrium between two conformers. However, aggregation did exist even at a low concentration of 1 microgram/ml. Aggregation also occurred without the conformational change as found at the initial stage or in Tris buffer, which suggested the absence of direct coupling between the conformational change and the aggregation. Changes of c.d. at 222 nm, expected to represent the main chain conformation, differed from those at 290 nm reflecting the environment of side chain chromophores. Time courses of three properties examined, c.d. at 222 nm, at 290 nm, and I90, always exhibited a lag in the case of metal-complexed species while the lag was not observed in the case of demetallized species, however. Lag became longer in c.d. but it became shorter in I90 as the protein concentration increased.
Circular Dichroism↗
Phosphoinositide-specific phospholipase C of a cloned mast cell line, mastocytoma P-815: purification and some characterization.
1. Multiple forms of phosphoinositide-phospholipase C (PLC) were isolated from mastocytoma; two cytosolic forms (cPLC-I, Mr 150,000; cPLC-II, Mr 110,000) and two membrane-associated forms (mPLC-I, Mr 85,000; mPLC-II, Mr 85,000). 2. Four PLC forms differently behaved in substrate specificity and effect of GTP-binding proteins.
Animals↗
[Growth of endometrial cancer and hormone].
Proliferation and differentiation of the normal endometrium are orderly regulated by female sex steroid hormones. In this connection, development and growth of endometrial cancer have also thought to be controlled in part by sex steroid hormones. Furthermore, some of the sex steroid hormones, progesterone, for example, are used as therapeutic agents in the management of endometrial cancer. The role of estrogen as a promotion factor of endometrial cancer is understood by unopposed estrogen hypothesis, and relative excess of estrogen unopposed by gestagen is regarded as an important factor for the development of endometrial cancer. High dose administration of gestagen has been used as a therapeutic agent of endometrial cancer over these three decades, and now the oral administration of medroxyprogesterone acetate (MPA) is mainstay, with response rate of approximately 30%. However, recently some cases with serious side effect, mainly thrombosis, have been reported. These cases should be regarded as a grave warning to easy usage of MPA. Therefore, the search for more effective and safe way for clinical application have to be requested; for example, clarification of the precise mechanism of anti-tumor effect of MPA on endometrial cancer or development of new hormonal therapeutic agents. Moreover, basic research in the field of cancer and hormone may create a new era in cancer therapy in the future.
Aged↗
[Superselective transcatheter-arterial-chemo-embolization in uterine cervical cancer].
Transcatheter-arterial-chemo-embolization (TACE) was performed on 7 cases of uterine cervical cancer from T1b to T3b. The purpose of this approach is to stop the uterine bleeding caused by malignancy and disrupt the cancer cells under the feeding arteries. TACE by Seldinger's method was undertaken selectively on both uterine arteries with suspension of Lipiodol, Adriamycin and Gelfoam. In each case, the bleeding stopped soon after the operation, and the lesion began to necrose within a few days. Though the primary effects were confirmed, complete remission could not be expected, so radiation therapy was performed. The longest survival among these patients was over 6 years. It should be noted that the other major advantage claimed for TACE is to decrease the dose of radiation.
Adult↗
[Recent progress in phospholipase C research in signal transduction].
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Amino Acid Sequence↗