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1.
The direct binding of porcine pancreatic phospholipase A2 and its zymogen to 1,2-bis(heptanylcarbamoyl)-rac-glycerol 3-sulfate was studied by optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. The zero-field splittings of the single Trp3 residue undergo significant changes upon binding of phospholipase A2 to lipid. Shifts in zero-field splittings, characterized mainly by a reduction of the E parameter from 1.215 to 1.144 GHz, point to large changes in the Trp3 local environment which accompany the complexing of phospholipase A2 with lipid. This may be attributed to Stark effects caused by the binding of a charged group near Trp3 in the enzyme-lipid complex. The cofactor, Ca2+, which is strongly bound to the enzyme active site, has an influence on the bonding, as reflected by smaller zero-field splitting shifts. A relatively small change in the Trp environment was observed for the interaction of the zymogen with lipid. 相似文献
2.
Changes in Targeting Efficiencies of Proteins to Plant Microbodies Caused by Amino Acid Substitutions in the Carboxy-terminal Tripeptide 总被引:7,自引:0,他引:7
Hayashi Makoto; Aoki Masahiro; Kondo Maki; Nishimura Mikio 《Plant & cell physiology》1997,38(6):759-768
It has been demonstrated that the carboxyl terminus of microbodyenzymes functions as a targeting signal to microbodies in higherplants. We have examined an ability of 24 carboxy-terminal aminoacid sequences to facilitate the transport of a cytosolic passengerprotein, ß-glucuroni-dase, into microbodies in greencotyledonary cells of trans-genic Arabidopsis. Immunoelectronmicroscopic analysis revealed that carboxy-terminal tripeptidesequences of the form [C/A/S/P]-[K/R]-[I/L/M] function as amicrobody-targeting signal, although tripeptides with prolineat the first amino acid position and isoleucine at the carboxylterminus show weak targeting efficiencies. All known micro-bodyenzymes that are synthesized in a form similar in size to themature molecule, except catalase, contain one of these tripeptidesequences at their carboxyl terminus. (Received April 14, 1997; Accepted April 8, 1997) 相似文献
3.
Teresa K. Aman Bruce A. Maki Thomas J. Ruffino Eileen M. Kasperek Gabriela K. Popescu 《The Journal of biological chemistry》2014,289(27):18805-18817
Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca2+ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca2+ permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca2+ conductance, because neither Na+ conductance nor Ca2+-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca2+ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca2+ permeability. 相似文献
4.
T Ryushi T Fukunaga K Yuasa H Nakajima 《European journal of applied physiology and occupational physiology》1990,60(1):44-48
The purpose of the present study was to investigate the influence of muscle fibre composition and stature on fractionated patellar reflex times in ten healthy untrained men (mean age: 23.3 years, SD 3.1; mass: 65.9 kg, SD 8.5; height: 172.3 cm, SD 5.3). Biopsies were taken from the right vastus lateralis muscle. Using staining for myofibrillar adenosine triphosphatase after pre-incubation at pH 4.3 and 4.6, muscle fibres were classified into slow twitch (ST), fast twitch, oxidative-glycolytic (FTa) and fast twitch, glycolytic (FTb) fibres. Total patellar reflex time (TRT) and its fractionated components--reflex latency (LAT) and reflex motor time (MT)--were obtained from the mean of ten trials in each subject whilst performing Jendrassik's maneuvre. The TRT, LAT and MT were 77.7 ms, SD 16.5, 23.4 ms, SD 1.3 and 54.2 ms, SD 16.3, respectively. The LAT was significantly correlated to the percentage number of ST (r = 0.758, P less than 0.05) and FTa fibres (r = -0.657, P less than 0.05), fast twitch:slow twitch ratio (r = -0.799, P less than 0.01) and to the height of the subjects (r = 0.901, P less than 0.001), whereas TRT and MT were not significantly correlated with either fibre types or the height of the subjects. From these results it can be concluded that the LAT during the patellar reflex is influenced by muscle fibre composition and the length of the sensory and/or motor nerve. 相似文献
5.
S Shoji J Ohnishi T Funakoshi K Fukunaga E Miyamoto H Ueki Y Kubota 《Journal of biochemistry》1987,102(5):1113-1120
The phosphorylation sites of myelin basic protein from bovine brain were determined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala- and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue. 相似文献
6.
Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase 总被引:1,自引:1,他引:0
Nobuhiro Inoue Takafumi Iwasa Kohji Fukunaga Yasuhiko Matsukado Eishichi Miyamoto 《Journal of neurochemistry》1987,48(3):981-988
Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme. 相似文献
7.
DNA binding of the mouse class II major histocompatibility CCAAT factor depends on two components. 总被引:8,自引:2,他引:6 下载免费PDF全文
A CCAAT-binding factor that recognizes a CCAAT sequence (Y box) located upstream of the major histocompatibility class II gene I-A beta has been partially purified. This CCAAT-binding factor was found to consist of two components, designated factors A and B, both of which were required for efficient binding to the DNA. Factor A had an apparent molecular size of 34 kilodaltons, and factor B had an apparent molecular size of 42 to 46 kilodaltons. 相似文献
8.
Triplet state properties of tryptophan residues in complexes of mutated Escherichia coli single-stranded DNA binding proteins with single-stranded polynucleotides. 下载免费PDF全文
Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed. 相似文献
9.
Tominaga Yoshito; Kuchitsu Kazuyuki; Katsuhara Maki; Tazawa Masashi; Miyachi Shigetoh 《Plant & cell physiology》1991,32(2):261-268
Rotational streaming of the cytoplasm including chloroplastswas induced by L-histidine, as well as by light, on the anticlinalface of leaf cells of Egeria densa. In the case of treatmentwith L-histidine some of the chloroplasts remained stationaryon the periclinal face of cells after rotational cytoplasmicstreaming was initiated. However, these chloroplasts were easilydislodged and translocated to the centrifugal end of the histidine-treatedcells by application of a centrifugal force that barely affectedthe location of chloroplasts in cells incubated in the darkwithout L-histidine. This result indicates that the anchoringof chloroplasts was weakened by L-histidine. Thus only the releaseof chloroplasts from anchoring was not enough for initiationof their streaming. The cytoplasmic pH (pHc) and vacuolar pH(pHv) were noninvasively monitored by in vivo 31P-nuclear magneticresonance (NMR) spectroscopy. Compared with the dark controlvalue, both illumination and treatment with L-histidine increasedthe pHc by 0.3 units. In contrast, pHv changed only a littlewith both illumination and treatment with L-histidine. Releaseof chloroplasts from anchoring and initiation of cytoplasmicstreaming are discussed in relation to the increase in pHc inducedby both light and L-histidine.
4 Present address: Department of Cell Biology, National Instituteof Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, 305Japan
5 Present address: Marine Biotechnology Institute Co., Ltd.,Head Office, 2-35-10 Hongo, Bunkyo-ku, Tokyo, 113 Japan (Received July 16, 1990; Accepted December 20, 1990) 相似文献
10.
Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane. 相似文献