The photosynthetic reaction center (RC) is the first membrane protein whose three-dimensional structure was revealed at the atomic level by X-ray crystallograph more than fifteen years ago. Structural information about RC made a great contribution to the understanding of the reaction mechanism of the complicated membrane protein complex. High-resolution structures of RCs from three photosynthetic bacteria are now available, namely, those from two mesophilic purple non-sulfur bacteria, Blastochloris viridis and Rhodobacter sphaeroides, and that from a thermophilic purple sulfur bacterium, Thermochromatium tepidum. In addition, a variety of structural studies, mainly by X-ray crystallography, are still being performed to give more detailed insight into the reaction mechanism of this membrane protein. This review deals with structural studies of bacterial RC complexes, and a discussion about the electron transfer reaction between RCs and electron donors is the main focus out of several topics addressed by these structural studies. The structural data from three RCs and their electron donors provided reliable models for molecular recognition in the primary step of bacterial photosynthesis.
In retroviral gene transduction, the efficiency of viral infection was reduced by the pro-teoglycans and some other materials secreted by the producer lines. In order to remove these inhibitors we have developed the rFN-CH-296-facilitated protocol. Because the rFN-CH-296 molecule has strong ability to bind a retroviral vector, rFN-CH-296 bound plates are utilized to wash out the unbound putative inhibitors present in a virus supernatant. The gene transduction efficiencies of human CD34*CD38- BMCs with a GALV-pseudotyped vector and the rFN-CH-296-facilitated protocol were compared with the protocol without a coating plate with CH-296, the mean gene transduction efficiencies being found to be 95. 5 and 71.1%, respectively.
An arginine-specific ADP-ribosyltransferase activity was detected in chicken gizzard smooth muscle, and the specific activity is highest in the membrane fraction. This trans-ferase is released from the membrane fraction by phosphatidylinositol-specific phospho-lipase C (PI-PLC), suggesting that it is a glycosylphosphatidylinositol (GPI)-anchored protein. When primary cultured gizzard smooth muscle cells (SMCs) were incubated with [Eadenylate-32P]NAD, several proteins were labeled. The labeling was inhibited by preincubation of the cells with PI-PLC, or by the addition of 4-arginine to the reaction, and was sensitive to hydroxylamine treatment. The activity of the transferase was main-tained in differentiated SMCs cultured with insulin, but was dramatically decreased concomitantly with cell dedifferentiation induced by serum or a specific PI3-Itinase LY294002. These results indicate that the GPI-anchored arginine-specific ADP-ribosyltransferase is expressed on the surface of differentiated SMCs and can modify several cell surface proteins. Our results also suggest that PI3-kinase is involved in the regulation of transferase activity during differentiation.
The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80°C of thermostable β-glycosidases from Sulfblobus solfataricus (SsβGly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl β-D-galactopyranoside or lactose (4-O-β-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, kNu/kwater, for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (kNu, M-1s-1) and water (kwater, s-1) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute ≈8 kJ mol-1 to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of SsβGly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol. The leaving group/nucleophile binding sites of SsβGly and CelB appear about 0.8 times as hydrophobic as n-octanol. Values of kNu/kwater for reactions of galactosylated SsβGly with ethanol and substituted derivatives of ethanol show no clear dependence on the pKa of the primary hydroxy group of these nucleophiles in the pKa range 12.4-16.0. The binding of phenol with the galactosylated enzyme intermediates of SsβGly and CeIB occurs in a form that is mainly nonproductive pertaining to β-galactoside synthesis. Neither enzyme catalyzes galactosyl transfer to azide ion. A model is proposed for the interaction of neutral nucleophiles at an extended acceptor site of the galactosylated en-zymes.
The present study was undertaken in an attempt to clarify the pathway by which hyperosmotic stress induces HB-EGF gene expression in rat aortic smooth muscle cells (RASMC). Hyperosmotic stress induced by a high concentration of glucose or mannitol resulted in an increase in HB-EGF mRNA level in a dose-and time-dependent manner. HB-EGF induction was blocked by curcumin, a c-jun/fos antisense oligonucleotide and a dominant-negative mutant of JNK1. Electrophoretic mobility shift assay also showed the involvement of AP-1 in HB-EGF gene expression by glucose. In addition, hyperosmotic stress induced rapid phosphorylation of Pyk2 in RASMC. TPA and calcium chelating agents (BAPTA-AM and EGTA) blocked Pyk2 phosphorylation and HB-EGF gene expression. Furthermore, HB-EGF gene expression and JNK activation by hyperosmotic stress were sensitive to PP2, an Src kinase-specific inhibitor. These findings indicate that hyperosmotic stress activates JNK via calcium-Pyk2 signaling cascades, which in turn induce HB-EGF gene expression.
In mouse, an oocyte-specific short isoform of DNA methyltransferase-1 (Dnmt1) lacking amino terminal 118 amino acid residues exists and plays a crucial role in maintaining the methylation state of imprinted genes during early embryogenesis [Howell et al. (2001) Cell 104, 829-838]. To address the question of whether or not Xenopus oocyte expresses such a short isoform, we raised monoclonal antibodies against the amino-terminal portion of Xenopus Dnmt1. Two of the isolated monoclonal antibodies, 3C6 and 4A8, were determined to recognize (1-32) and (115-126) of Xenopus Dnmt1, respectively. The amounts of Dnmtl in Xenopus eggs were determined to be similar, 10.0 ± 2.5, 8.0 ± 0.8, and 8.2 ± 0.2 ng per egg with monoclonal antibodies 3C6 and 4A8, and polyclonal antibodies, respectively. This indicated that Dnmt1 in Xenopus mature eggs had an identical amino-terminal sequence to the amino acid sequence deduced from the cDNA. Together with the fact that Dnmtl in All cells immuno-reacted with all the monoclonal antibodies isolated and with the polyclonal antibodies, we concluded that Dnmtl expressed in Xenopus mature eggs possesses an identical amino-terminal sequence to that in somatic cells. Immuno-purified Xenopus Dnmt1 in mature eggs showed similar specific activity to that in proliferating A6 cells and that of mouse recombinant Dnmt1.
In this work we present a method for ultra-fine patterning of primary culture neuron cell growth, which is compatible for scanning near-field optical atomic forcemicroscopy (SNOAM) analysis. SNOAM uses near-field optics to break the fundamental diffraction limit imposed on normal microscopy. SNOAM can achieve sub-100 nm optical resolutions, but requires transparent, open substrates. The ability to do physiological measurements on patterns of neurons, combined with ultra high resolution optical and fluorescent analysis, is useful in the study of long-term potentiation. The patterning method consists of chemical guidance with an element of physical confinement and allows for ultra-fine patterning of neural growth on transparent glass substrates. Substrates consist of microfabricated perfluoropolymer barrier structures on glass. Poly-L-lysine was selectively deposited using a silicone-based microfluidic stencil aligned to the perfluoropolymer/glass substrate. Primary culture neurons were extracted from 8-dayold chicks and grown for 3 days to form good networks. This patterning system shows very specific growth with patterning separations down to the level of individual neurites. Fluorescent imaging was carried out on both cell viability during growth and immuno-tagged microtubule-associated proteins on the neurites. Neurons inside the patterned structures were imaged and analyzed with a tapping mode SNOAM.neuron, patterning, SPM, SNOM, SNOAM.
From the aquatic bacterium Rhodococcus equi strain S420, we isolated a substance that strongly binds to influenza viruses. Structural analyses revealed that it is a unique type of phosphatidylinositol (Ptdlns) bearing a branched-chain fatty acid (14-methyloctadecanoic acid). In a TLC/virus-binding immunostaining assay, this Ptdlns bound to all subtypes of hemagglutinin (HA) of influenza A viruses tested, isolated from humans, ducks and swine, and also to human influenza B viruses. Furthermore, the Ptdlns significantly prevented the infection of MDCK cells by influenza viruses, and also inhibited the virusmediated hemagglutination and low pH-induced hemolysis of human erythrocytes, which represents the fusogenic activities of the viral HA. We also used purified hemagglutinin instead of virions to examine the interaction between viral HA and Ptdlns, showing that the Ptdlns binds to hemagglutinin. These findings indicate that the inhibitory mechanism of Ptdlns on the influenza virus infection may be through its binding to viral HA spikes and host cell endosomal/lysosomal membranes, which are mediated by the function of viral HA.
The Maf recognition element (MARE) is regulated by both activators and repressors. Bachl and Bach2 repress MARE-dependent transcription by forming heterodimers with Maf-related oncoproteins. In order to gain an understanding of the regulation of bachl gene expression, we analyzed the structure of the mouse bachl gene. Comparison of the exon-intron structure of the bachl gene with those of other NF-E2-related genes indi-cated that bachl and bach2 constitute an evolutionarily distinct subfamily among bZip factors. The bachl promoter region contains two GC boxes that are important for its basal activity and are bound by Spl in K562 cell extracts. In addition, we found an evo-lutionarily conserved MARE-like element located downstream of the transcription initi-ation site. Deletion of this element resulted in a higher promoter activity in K562 cells. Bachl trans-activated its own promoter depending on the presence of the MARE-like element in co-transfection assays. However, Bachl did not bind to the MARE-like ele-ment in electrophoretic mobility shift assays (EMSA). These results suggest that Bachl activates its own promoter indirectly by inhibiting the putative repressor. Such a posi-tive feedback regulation by the repressor Bachl may play an important role in main-taining the expression of Bachl while consolidating repression of other genes with MARE.
Melittin-induced membrane fusion between neutral and acidic phospholipids was exam-ined in liposome systems with a high-sensitivity differential scanning calorimeter. Mem-brane fusion could be detected by calorimetric measurement by observing thermograms of mixed liposomal lipids. The roles of hydrophobic and electrostatic interactions were investigated in membrane fusion induced by melittin. Melittin, a bee venom peptide, is composed of a hydrophobic region including hydrophobic amino acids and a positively charged region including basic amino acids. When phosphatidylcholine liposomes were prepared in the presence of melittin, reductions in the phase transition enthalpies were observed in the following order; dimyristoylphosphatidylcholine (DMPC)>dipalmi-toylphosphatidylcholine (DPPC)>distearoylphosphatidylcholine (DSPC)>dielaidoyl-phosphatidylcholine (DEPC). The plase transition enthalpy of an acidic phospholipid, dipalmitoylphosphatidylserine (DPPS), was raised by melittin at low concentrations, then reduced at higher concentrations. DPPC liposomes prepared in melittin solution were fused with DPPS liposomes when the liposomal dispersions were mixed and incu-bated. Similar fusion was observed between dipalmitoylphosphatidylcholine and di-myristoylphosphatidic acid (DMPA) liposomes. These results indicate that a peptide in-cluding hydrophobic and basic regions can mediate membrane fusion between neutral and acidic liposomes by hydrophobic and electrostatic interactions.
Plasmid pSC101 encodes a 37. 5 kDa Rep (RepA) protein, which binds to three 21-base repeats (DR-1, DR-2, and DR-3) in the replication origin region (ori) of the plasmid to ini-tiate replication. Rep also binds to two palindromic sequences (IR-1 and IR-2) which overlap the rep promoter. The binding of Rep to IR-2 represses the production of Rep itself. It is highly likely that the balance of these functions of Rep plays a major role in controlling the copy number of pSC101. In this study, we developed a positive-selection system for replication-deficient mutants of the initiator protein. This system can be applied to the study of other replication systems by changing ori and rep of pSC101 to the corresponding genes. Thirty-four replication-deficient (Ini-) mutants were isolated with this system, and analyzed as to the relation between the structure and function of the Rep protein. Seventeen of these 34 Ini-mutants were found to lack auto-repressor activity as well as initiator activity. DNA sequence analysis showed that one-third (from the C-terminus) of Rep is dispensable for the auto-repressor activity, while the initiator activity seems to require the whole protein.
Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the α and β chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recog-nized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other mono-meric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggre-gation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 _??_m in diameter), medium-sized and large aggregates (25 to 70 _??_m in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maxi-mal number on either treatment with ADP alone or monomeric disintegrin/ADP. How-ever, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without plate-let activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dis-sociation.
In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging noncanonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearotherinophilus TyrRS. Among these mutants, one with the replacement of tyrosine at position 43 by glycine, “Y43G, ” was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (kcat/Km) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions. This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro.
Previously we isolated and characterized a membrane-bound, arginine-specific serine protease from pig intestinal mucosa [J. Biol. Chem. 269, 32985-32991 (1994)]. For further characterization of this type of enzyme, we cloned a cDNA from rat intestinal mucosa encoding the precursor of a similar protease. The partial amino acid sequences deter-mined for the pig enzyme were found to be shared almost completely by the rat enzyme. The serine protease domain of the rat enzyme, heterologously expressed in Escherichia coli, specifically cleaved Arg (or Lys)-X bonds with a marked preference for Arg-Arg or Arg-Lys, similar to the pig enzyme. The mRNA for the rat enzyme was shown to be distributed mainly in intestine, and the enzyme was detected in the duodenal mucosa as a 70 kDa protein. Immunohistochemical analysis of the small intestinal tissue showed that the enzyme is localized mainly on brushborder membranes.
Bdellin-KL is a trypsin-plasmin inhibitor from Hirudo nipponia, whose N-terminal sequence was identified as a non-classical Kazal-type. A cDNA clone encoding the inhibitor was isolated by reverse transcription-PCR and 5' rapid amplification of cDNA ends. The cDNA showed an open reading frame of 155 amino acids comprising one signal peptide and two separated domains. The C-terminal domain consists of distinct internal repeats, including HHEE and HHDD. The bdellin-KL sequence, from the constructed genomic library of Korean leech, was determined for the 2109 bases comprising the open reading frame and flanking regions (3' and 5'). The promoter region contains potential regulatory sequence motifs, including TATA, CAAT, and GC boxes. To characterize the properties of each domain, an N-terminal fragment was prepared by limited proteolysis of the intact protein. The inhibitory activity of the region was as potent as that of the intact protein. This suggests that the compact domain plays an important part in the inhibitory action of bdellin-KL. The C-terminal domain was revealed to have binding affinity to ions such as Ca2+, Zn2+, Fe3+, and Fe2+. without an influence on the inhibitory activity. This study demonstrates that bdellin-KL may be a novel bifunctional protein with two distinct domains.
To elucidate the roles of tryptophan residues in the structure, stability, and function of Escherichia coli dihydrofolate reductase (DHFR), its five tryptophan residues were re-placed by site-directed mutagenesis with leucine, phenylalanine or valine (W22F, W22L, W3OL, W47L, W74F, W74L, W133F, and W133V). Far-ultraviolet circular dichroism (CD) spectra of these mutants reveal that exciton coupling between Trp47 and Trp74 strongly affects the peptide CD of wild-type DHFR, and that Trp133 also contributes appreciably. No additivity was observed in the contributions of individual tryptophan residues to the fluorescence spectrum of wild-type DHFR, Trp74 having a dominant effect. These single-tryptophan mutations induce large changes in the free energy of urea unfolding, which showed values of 1. 79-7. 14 kcal/mol, compared with the value for wild-type DHFR of 6. 08 kcal/mol. Analysis of CD and fluorescence spectra suggests that thermal unfolding involves an intermediate with the native-like secondary structure, the disrupted Trp47-Trp74 exciton coupling, and the solvent-exposed Trp30 and Trp47 side chains. All the mutants except W22L (13%) retain more than 50% of the enzyme activity of wild-type DHFR. These results demonstrate that the five tryptophan residues of DHFR play important roles in its structure and stability but do not crucially affect its enzymatic function.
To identify lung lamellar body (LB)-binding proteins, the fractions binding to LB-Se-pharose 4B in a Ca2+-dependent manner from the lung soluble fractions were analyzed with Mono Q column. Four annexins (annexins III, IV, V, and VIII) were identified by partial amino acid sequence analyses as the LB-binding proteins in the lung soluble fractions. A control experiment using phospholipid (phosphatidylserine/phosphatidylg-lycerol/phosphtidylcholine) liposome-Sepharose 4B revealed that annexins III, IV and V were the Ca2+-dependent proteins binding to the column in the lung soluble fractions, while annexin VIII was not detected. Thus, annexin VIII might preferentially bind to LB. On the other hand, the only Ca2+-dependent LB-binding protein identified in the bron-choalveolar lavage fluids was annexin V. It was further demonstrated that annexin V was secreted by isolated alveolar type II cells from rats and that the secretion was stimulated by the addition of phorbol ester (PMA), a potent stimulator of surfactant secretion. The PMA-dependent stimulation of annexin V was attenuated by preincubation with surfactant protein-A (SP-A), a potent inhibitor of surfactant secretion. As LB is thought to be an intracellular store of pulmonary surfactant, which is secreted by alveolar type II cells, annexin V is likely to be secreted together with the lamellar body.
Retinoylation (retinoic acid acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro and in tissues in vivo. The widespread occurrence of retinoylation suggests that it may play a role in many effects of retinoic acid (RA) on cells. One metabolic pathway for retinoylation involves the intermediate formation of retinoyl-CoA and subsequent transfer and covalent binding of the retinoyl moiety to protein. However, such reactions are not well known. To gain further insight into retinoylation, we studied the synthesis of retinoyl-CoA, the first step in this multi-stage process. The formation of [3H]-retinoyl-CoA was determined in incubation mixtures containing rat liver extract, [3H]-RA, ATP, CoA, and MgCl2. No retinoyl-CoA was formed in the presence of boiled extract, or in the absence of ATP, CoA, or MgC12 (a divalent cation). A greater amount of retinoyl-CoA was obtained from microsomal fractions of rat liver than from other subfractions. The presence of retinoyl-CoA was also detected in extracts prepared from rat testis, kidney, brain, spleen, and pancreas. The level of retinoylation in various tissue extracts was related directly to the amount of retinoyl-CoA formed. Vmax and Km, values for RA in the formation of liver retinoyl-CoA were estimated to be 1.0 x 10-4 μimol/minimg protein and 24 nM, respectively. Synthesis of retinoyl-CoA was suppressed by fatty acids and fatty acyl-CoAs. These results indicate that ATP-dependent generation of retinoyl-CoA occurs in rat tissues and may play a significant physiological role in RA actions mediated by retinoylation.
Glycogen debranching enzyme was partially purified from bovine brain using a substrate for measuring the amylo-1, 6-glucosidase activity. Bovine cerebrum was homogenized, followed by cell-fractionation of the resulting homogenate. The enzyme activity was found mainly in the cytosolic fraction. The enzyme was purified 5000-fold by ammonium sulfate precipitation, anion-exchange chromatography, gel-filtration, anion-exchange HPLC, and gel-permeation HPLC. The enzyme preparation had no α-glucosidase or α-amylase activities and degraded phosphorylase limit dextrin of glycogen with phos-phorylase. The molecular weight of the enzyme was 190000 and the optimal pH was 6.0. The brain enzyme differed from glycogen debranching enzyme of liver or muscle in its mode of action on dextrins with an α-1, 6-glucosyl branch, indicating an amino acid sequence different from those of the latter two enzymes. It is likely that the enzyme is involved in the breakdown of brain glycogen in concert with phosphorylase as in the cases of liver and muscle, but that this proceeds in a somewhat different manner. The enzyme activity decreased in the presence of ATP, suggesting that the degradation of brain glycogen is controlled by the modification of the debranching enzyme activity as well as the phosphorylase.