Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle.
The ratio of the absorption of myosin S at 280 nm to that at 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/105 g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KCl and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A.
Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per 105 g of protein as horse and rabbit skeletal myosin A (about 8 moles/105 g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline.
Ca2+-ATPase of myosin S in the presence of 0.5 M KCl and Mg2+-ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca2+-ATPase in the presence of 0.05 M KCl were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25°, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N-ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca2+-ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca2+-ATPase during the course of the reaction of sulfhydryl groups of myosin with N-ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A. 相似文献
In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale. 相似文献
To evaluate the genetic susceptibility to metabolic disorders induced by high fructose diet, we investigated the metabolic characteristics in 10 strains of inbred mice and found that they were separated into CBA and DBA groups according to the response to high fructose diet. The hepatic mRNA expression of the sterol regulatory element-binding protein-1 (SREBP-1) in CBA/JN was remarkably enhanced by high fructose diet but not in DBA/2N. Similar results were observed in primary hepatocytes after exposure to fructose. The nucleotide sequence at -468 bp from the putative starting point of the SREBP-1c gene was adenine in the DBA group while it was guanine in the CBA group. In hepatocytes from CBA/JN, the activity of CBA-SREBP-1c promoter was significantly increased by 2.4- and 2.2-fold, in response to 30 mm fructose or 10 nm insulin, respectively, whereas the activity of DBA-SREBP-1c promoter responded to insulin but not to fructose. In hepatocytes from DBA/2N, both types of SREBP-1c promoter activities in response to insulin were attenuated. Furthermore, electrophoretic mobility shift assay revealed an unidentified nuclear protein bound to the oligonucleotides made from the region between -453 to -480 bp of the SREBP-1c promoter of CBA/JN but not to the probe from DBA/2N. Thus, in DBA/2N, the reduced mRNA expression of SREBP-1 after fructose refeeding appeared to associate with two independent mechanisms, 1). loss of binding of unidentified proteins to the region between -453 to -480 bp of the SREBP-1c promoter and 2). impaired insulin stimulation of SREBP-1c promoter activity. 相似文献
The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCdelta as well as its kinase-negative mutant in MeWo cells, which contain PKCalpha but lack PKCbeta. PLD activity was not affected by PKCdelta in G361 cells, which have PKCbeta but are deficient in PKCalpha. In MeWo cells introduced by PKCalpha and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCdelta in addition to PKCalpha and PLD1, TPA treatment increased the association of PKCdelta and PLD1, while it attenuated the association of PKCalpha and PLD1. These results indicate that PKCdelta inhibits TPA-induced PLD1 activation mediated by PKCalpha through the association with PLD1. 相似文献
Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation. 相似文献
Since the rat is an atherosclerosis-resistant species, the study of atherosclerosis using rats is limited. The present study was undertaken to develop an atherosclerotic model in rats, to investigate the effect of nitric oxide (NO) inactivation and hyperlipidemia, and to evaluate the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor, on NO inactivation and on hyperlipidemia-induced changes in the cardiovascular system. Four-month-old male spontaneously hypertensive hyperlipidemic rats (SHHR) and Sprague-Dawley (SD) rats were used to study 1) the effect of the period of treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/L) on high fat diet (HFD)-treated SHHR and SD rats, and 2) the effect of pitavastatin (Pit, 0.3 mg/kg/day) on the changes in the aorta of L-NAME- and HFD-treated SHHR and SD rats. L-NAME administration for 1 month then HFD feeding for 2 months markedly increased the deposition of lipids and the thickness of the endothelium in SHHR. Continuous L-NAME treatment with HFD produced severe injury and stripped of endothelium in both strains. The plasma total cholesterol of L-NAME + HFD-treated and L-NAME + HFD + Pit-treated SHHR was significantly higher than that of control SHHR. Lipid deposition, however, was comparatively less in the aorta of L-NAME + HFD + Pit-treated SHHR. The concentration of cholesterol in the aorta of control SHHR was significantly lower than that in the aorta of L-NAME + HFD-treated SHHR, whereas that of L-NAME + HFD + Pit-treated SHHR was the same as that in control SHHR. These data indicated that Pit blocked lipid deposition in the aorta of L-NAME + HFD treated SHHR without changing plasma lipid profiles. In conclusion, NO inactivation and HFD induce lipid deposition in the endothelium, and the HMG-CoA reductase inhibitor blocks the deposition in SHHR. 相似文献
Crosslinking of multivalent antigen bound IgE transduces FcepsilonRI mediated signaling cascades, which activate nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, and these are critical elements for degranulation in mast cells. We cloned a novel adaptor molecule, signal transducing adaptor protein (STAP)-2 containing PH and SH2-like domains as a c-fms interacting protein. STAP-2 was identical to a recently cloned adaptor molecule, BKS, a substrate of BRK (breast tumor kinase) tyrosine kinase, although its function is still unknown. To examine a novel function of STAP-2/BSK, we expressed STAP-2/BSK or its mutants in rat basophilic leukemia RBL-2H3 cells. Overexpression of STAP-2/BSK resulted in a suppression of FcepsilonRI-mediated calcium mobilization and degranulation. FcepsilonRI-induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) but not Syk was significantly suppressed in these cells. Furthermore, STAP-2/BSK associated with PLC-gamma in vivo. These data indicate that STAP-2/BSK negatively controls the FcepsilonRI-mediated calcium mobilization and degranulation by direct modulation of tyrosine phosphorylation of PLC-gamma. 相似文献