Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.     

Inhibitor of interferon-induced double-stranded RNA-dependent protein kinase and its relevance to alteration of cellular protein kinase activity level in response to external stimuli.

In FL cells, interferon (IFN)-induced dsRNA-dependent protein kinase (PK-I) was found to be present in a form complexed with a potent inhibitor of its dsRNA-dependent activation. The inhibitor was readily dissociated from PK-I by DEAE-cellulose chromatography to yield a dsRNA-responsive PK-I. The inhibitor was also dissociated easily from PK-I by gel filtration through Sephacryl S-200. The apparent molecular mass of the inhibitor as estimated by gel filtration was more than 160 kilodaltons. Activity of the inhibitor was decreased on IFN treatment for 8.5 hr or on Sindbis virus infection with concomitant increase in the amount of dsRNA-activatable form of PK-I. This result implies that the inhibitor may be one of the regulatory factors of cellular PK-I activity. Longer IFN treatment (24 hr) led to recovery of the inhibitor activity, but it was overridden by an extensive net synthesis of the PK-I protein.     

Characterization of Gd3+ and Tb3+ binding sites on Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum

Interaction between Gd3+ and Tb3+ ions and Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was studied. Three classes of lanthanide-ion binding sites with different affinities were distinguished. Binding of Gd3+ to the site with the highest affinity seemed to occur at less than 10(-6)M free Gd3+ and resulted in severe inhibition of ATPase activity. The reaction rates of both E-P formation and decomposition in the forward direction were inhibited in parallel with this binding, whereas ADP-dependent decay of E-P in the backward direction was not. At these Gd3+ concentrations, Ca2+-binding to the transport site was not inhibited. Binding of Gd3+ and Tb3+ to the Ca2+-transport site did occur, but more than 10(-5)M free Gd3+ or Tb3+ was required for effective competition with Ca2+ for that site. Gd3+ bound to the transport site in place of Ca2+ did not activate the E-P intermediate formation. Addition of 10(-1)M Tb3+ to a suspension of sarcoplasmic reticulum membranes resulted in marked enhancement of Tb3+ fluorescence, which is due to an energy transfer from aromatic amino acid residues of ATPase to Tb3+ ions bound to the low affinity site of the enzyme. Gd3+ and Mn2+ competed with Tb3+ for that site, but Ca2+, Zn2+, and Cd2+ did not.     

Alteration of opioid receptors in seizure-susceptible El mouse brain

The distribution density of opioid receptors in the brain of El mice (seizure-susceptible strain) was examined to determine the relation between seizures and the opioid system. Saturation curves and Scatchard plots of [³H]2-d-alamine-5-d-leucine enkephalin binding revealed that the opioid delta receptor density in adult El mice during interictal periods was significantly increased in the cerebral cortex, hippocampus, and septal area. It was further shown that the concentration of such receptors in 25-day-old El mice that had no seizures was also significantly increased in the hippocampus and septal area, with no changes in apparent affinities, as compared with in the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). Such up-regulation of opioid receptors in the El mouse brain could result from deficits in endogenous opioid peptides, which could be associated with the pathogenesis of seizure diathesis in the El mouse.     

Nitric Oxide Induces Phytoalexin Accumulation in Potato Tuber Tissues

We investigated whether nitric oxide (NO) radical could inducephytoalexin production. Treatment of potato tuber tissues with1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC-18), anNO-releasing compound, induced the accumulation of the potatophytoalexin rishitin. This induction was inhibited by carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO), an NO-specific scavenger, or by Tiron, a radicalscavenger, suggesting a phytoalexin inducing activity for NO. (Received December 7, 1995; Accepted January 4, 1996)     

Immobilization of nucleoside diphosphatase at its allosteric site using immobilized derivatives of pyridoxal 5'-phosphate

3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH₄. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl₂ was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.     

Purification of interferon from mouse Ehrlich ascites tumor cells

Interferon production was induced in mouse Ehrlich ascites tumor cells by infection with Newcastle disease virus. The interferon produced was purified by precipitation with ammonium sulfate, chromatography on carboxymethyl-Sephadex, treatment with blue dextran and polyethylene glycol, gel filtration on Bio-Gel P-60 and Bio-Gel P-200, chromatography on phosphocellulose, isoelectric focusing, and chromatography on octyl-Sepharose. The specific activity of the product was 1.6 x 10(9) NIH mouse interferon reference standard units/mg of protein. Electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate indicated that the apparent molecular weight of the interferon-active material ranged from 25,000 to 35,000. As revealed by staining the gels with Coomassie brilliant blue, the interferon activity co-migrated with the major, broad protein band. Minor, stainable bands of proteins were free of interferon activity and their apparent molecular weight was smaller than 12,000.     

Antibody against neurofibromatosis type 1 gene product reacts with a triton-insoluble GTPase activating protein toward ras p21

Cellular fractionation of GTPase activating protein (GAP) activity using bovine cerebral cortex revealed that about half of GAP activity was found in membrane fraction. GAP activity of membrane was not solubilized with 0.5% (v/v) triton X-100 and was immunoprecipitated with antibody against carboxy-terminus of neurofibromatosis type 1 (NF1) gene product. In contrast, soluble GAP activity was precipitated with antibody against GAP but not with anti-NF1. These results suggest that NF1 gene product is a GTPase activating protein toward ras p21 with completely different intracellular distribution from that of GAP.     

Influence of surface anesthesia on the pressure pain threshold measured with different-sized probes

Transcutaneous pressure with pressure probes of arbitrary diameters have been commonly used for measuring the threshold and magnitude of muscle pain, yet this procedure lacks scientific validation. To examine the valid probe dimensions, we conducted physiological experiments using 34 human subjects. Pin-prick pain, pressure pain threshold (PPT) to pressure probes of various diameters, heat pain threshold, and electrical pain threshold of deep tissues were measured before and after application of surface lidocaine anesthesia to the skin surface over the brachioradial muscle in a double-blinded manner. The anesthesia neither affected PPT with larger probes (diameters: 1.6 and 15?mm) nor increased electric pain threshold of deep structures, whereas it diminished pain count in pin-prick test and PPT with a 1.0?mm diameter probe, suggesting that mechanical pain thresholds measured with 1.6 and 15?mm probes reflect the pain threshold of deep tissues, possibly muscle. Pain thresholds to heat did not change after application of the anesthesia. These results suggest that larger pressure probes can give a better estimation of muscular pain threshold.