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Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.

We examined the role of cyclic ADP-ribose (cADP-ribose) as a second messenger downstream of adrenergic receptors in the heart after excitation of sympathetic neurons. To address this question, ADP-ribosyl cyclase activity was measured as the rate of [(3)H]cADP-ribose formation from [(3)H]NAD(+) in a crude membrane fraction of rat ventricular myocytes. Isoproterenol at 1 microM increased ADP-ribosyl cyclase activity by 1.7-fold in ventricular muscle; this increase was inhibited by propranolol. The stimulatory effect on the cyclase was mimicked by 10 nM GTP and 10 microM guanosine 5'-3-O-(thio)triphosphate, whereas 10 microM GTP inhibited the cyclase. Cholera toxin blocked the activation of the cyclase by isoproterenol and GTP. The above effects of isoproterenol and GTP in ventricular membranes were confirmed by cyclic GDP-ribose formation fluorometrically. These results demonstrate the existence of a signal pathway from beta-adrenergic receptors to membrane-bound ADP-ribosyl cyclase via G protein in the ventricular muscle cells and suggest that increased cADP-ribose synthesis is involved in up-regulation of cardiac function by sympathetic stimulation.

The "switch I" region (Asp30-Asp38) of the Ras protein takes remarkably different conformations between the GDP- and GTP-bound forms and coincides with the so-called "effector region." As for a region on the C-terminal side of switch I, the V45E and G48C mutants of Ras failed to promote neurite outgrowth of PC12 cells (Fujita-Yoshigaki, J., Shirouzu, M., Koide, H., Nishimura, S., and Yokoyama, S. (1991) FEBS Lett. 294, 187-190). In the present study, we performed alanine-scanning mutagenesis within the region Lys42-Ile55 of Ras and found that the K42A, I46A, G48A, E49A, and L53A mutations significantly reduced the neurite-inducing activity. This is an effector region by definition, but its conformation is known to be unaffected by GDP-->GTP exchange. So, this region is referred to as a "constitutive" effector (Ec) region, distinguished from switch I, a "switch" effector (Es) region. The Ec region mutants exhibiting no neurite-inducing activity were found to be correlatably unable to activate mitogen-activated protein (MAP) kinase in PC12 cells. Therefore, the Ec region is essential for the MAP kinase activation in PC12 cells, whereas mutations in this region only negligibly affect the binding of Ras to Raf-1 (Shirouzu, M., Koide, H., Fujita-Yoshigaki, J., Oshio, H., Toyama, Y., Yamasaki, K., Fuhrman, S. A., Villafranca, E., Kaziro, Y., and Yokoyama, S. (1994) Oncogene 9, 2153-2157).

Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.

The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids. In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA. In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I. In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction. Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA. This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed.

Cyclic ADP-ribose (cADP-ribose) is an endogenous modulator of ryanodine-sensitive Ca2+ release channels. An unsolved question is whether or not cADP-ribose mediates intracellular signals from hormone or neurotransmitter receptors. The first step in this study was to develop a TLC method to measure ADP-ribosyl cyclase, by which conversion of [3H]NAD+ to [3H]cADP-ribose was confirmed in COS-7 cells overexpressing human CD38. A membrane fraction of NG108-15 neuroblastoma x glioma hybrid cells possessed ADP-ribosyl cyclase activity measured by TLC. Carbamylcholine increased this activity by 2.6-fold in NG108-15 cells overexpressing m1 or m3 muscarinic acetylcholine receptors (mAChRs), but inhibited it by 30-52% in cells expressing m2 and/or m4 mAChRs. Both of these effects were mimicked by GTP. Pretreatment of cells with cholera toxin blocked the activation, whereas pertussis toxin blocked the inhibition. Application of carbamylcholine caused significant decreases in NAD+ concentrations in untreated m1-transformed NG108-15 cells, but an increase in cholera toxin-treated cells. These results suggest that mAChRs couple to ADP-ribosyl cyclase within cell membranes via trimeric G proteins and can thereby control cellular function by regulating cADP-ribose formation.

Ras proteins have multiple effectors of distinct structures that do not share significant structural homology at their Ras interaction sites. To prove possible differences in their recognition mechanisms of Ras, we screened 44 human Ha-Ras proteins carrying mutations in the effector region and its flanking sequences for interaction with human Raf-1, Schizosaccharomyces pombe Byr2, and Saccharomyces cerevisiae adenylyl cyclase. The Ras binding specificities were largely shared between Raf-1 and Byr2 although Ras mutants, Y32F, T35S, and A59E, had their affinities for Byr2 selectively reduced. The only exception was Ras(D38N), which lost the ability to bind Raf-1 while retaining the activity to bind Byr2 and complement the Byr2- phenotype of S. pombe. On the other hand, adenylyl cyclase had quite distinct requirements for Ras residues; mutations P34G and T58A selectively abolished the ability to bind and activate it without considerably affecting the interaction with Raf-1 and Byr2. Y32F mutant, whereas losing the ability to activate Raf-1 and Byr2, could activate adenylyl cyclase efficiently. In addition, V45E mutation was found to impair the ability of Ras to activate both Raf-1 and adenylyl cyclase without significantly affecting the binding affinities for them. These results demonstrate that significant differences exist in the recognition mechanisms by which the three effector molecules associate with Ras and suggest that a region of Ras required for activation of the effectors in general may exist separately from that for binding the effectors.

Triolein particles stabilized with egg yolk phosphatidylcholine monolayer were prepared with two different diameters: 26.7 +/- 3.9 and 229 +/- 80 nm. All the phosphatidylcholine molecules in those particles were readily digested by phospholipase A2 while only the molecules in the outer leaflet of phosphatidylcholine unilamellar vesicles were hydrolyzed under the same conditions. Binding of human plasma apolipoproteins A-I, A-II, C-II, and C-III2 to the particles was studied by two independent techniques: (i) rapid gel permeation chromatography and (ii) ultracentrifugation. All four apolipoproteins bound to the small and large particles in a saturable manner without altering their gross structure, and were displaced by equivalent molecules. The dissociation constant of apolipoprotein A-I for the large particle was 3.17 X 10(-6) M and 4.24 X 10(-6) M by methods (i) and (ii), respectively. These values were more than 10-fold greater than those for the small particles (2.0 X 10(-7) and 1.6 X 10(-7) M, respectively). In contrast, apolipoproteins A-II, C-II, and C-III2 bound to the large particles as strongly as to the small particles with dissociation constants of 2.4-6.8 X 10(-7), 4.5-10.7 X 10(-7), and 5.3-10.7 X 10(-7) M, respectively. The maximum binding level was of a similar order for each of the four apolipoproteins with both lipid particles when they were compared on the basis of amino acids per phospholipid. These results suggest that the apolipoproteins share common binding sites on the lipid particles, and are consistent with the characteristic distribution of apolipoproteins A-I and C among various classes of lipoproteins in plasma.

A minor species of isoleucine tRNA (tRNA(minor Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor Ile)) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6) A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the epsilon amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(beta-D-ribofuranosyl)pyrimidinium (“lysidine”), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

Methionyl-tRNA synthetase (MetRS, 2 X 75 kDa) was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB8. The polypeptide chain of MetRS was cleaved by limited digestion with trypsin into four domains: T1 (29 kDa), T2 (23 kDa), T3 (14.5 kDa), and T4 (7.5 kDa), which were aligned in that order. MetRS was also cleaved into similar fragments with a variety of other proteases. Domains T1, T2, T3, and T4 were isolated by column chromatography. "Tandem domain" T1-T2 (56 kDa) is fully active in the aminoacylation of tRNA and is further cleaved with trypsin into domains T1 and T2. Domain T1 is the smallest aminoacylation unit so far reported. Domain T2 (enzymatically inactive) interacts with tRNAMetf, as found by UV-induced cross-linking. Isolated domain T3 forms a dimer and is responsible for the dimer assembly of two protomers in MetRS. Domain T4 is a flexible tail of MetRS. These domains, in particular T1 and T2, will be important for detailed structure analyses in relation to aminoacylation activity.

The Ras and Rap1A proteins can bind to the Raf and RalGDS families. Ras and Rap1A have Glu and Lys, respectively, at position 31. In the present study, we analyzed the effects of mutating the Glu at position 31 of the c-Ha-Ras protein to Asp, Ala, Arg, and Lys on the interactions with Raf-1 and RalGDS. The Ras-binding domain (RBD) of Raf-1 binds the E31R and E31K Ras mutants less tightly than the wild-type, E31A, and E31D Ras proteins; the introduction of the positively charged Lys or Arg residue at position 31 specifically impairs the binding of Ras with the Raf-1 RBD. On the other hand, the ability of the oncogenic RasG12V protein to activate Raf-1 in HEK293 cells was only partially reduced by the E31R mutation but was drastically impaired by the E31K mutation. Correspondingly, RasG12V(E31K) as well as Rap1A, but not RasG12V(E31R), exhibited abnormally tight binding with the cysteine-rich domain of Raf-1. On the other hand, the E31A, E31R, and E31K mutations, but not the E31D mutation, enhanced the RalGDS RBD-binding activity of Ras, indicating that the negative charge at position 31 of Ras is particularly unfavorable to the interaction with the RalGDS RBD. RasG12V(E31K), RasG12V(E31A), and Rap1A stimulate the RalGDS action more efficiently than the wild-type Ras in the liposome reconstitution assay. All of these results clearly show that the sharp contrast between the characteristics of Ras and Rap1A, with respect to the interactions with Raf-1 and RalGDS, depends on their residues at position 31.

Erythrocyte was found lacking in reactivity to lipid-free apolipoproteins to generate pre-beta-high density lipoprotein (HDL) with the cellular lipid and, therefore, was used to study cellular cholesterol efflux to plasma lipoproteins exclusively by a nonspecific exchange mechanism. Over the range of hematocrit from 1-20% (cellular cholesterol pool of 2.5 micrograms per 250 microliters), the fractional rate of cellular cholesterol efflux to lipoprotein was constant, and, therefore, absolute efflux rate was a linear function of the hematocrit of this range. In the absence of lecithin:cholesterol acyltransferase (LCAT), the cholesterol influx rate from lipoproteins was equal to the efflux rate from erythrocyte resulting in no net transfer of cholesterol, with either HDL or low density lipoprotein. In the presence of LCAT in the mixture of HDL and erythrocyte, cholesterol was esterified exclusively in HDL regardless of the origin. When the hematocrit was low and efflux of cellular cholesterol was slower than cholesterol esterification, the esterification of cell-originating cholesterol did not directly enhance the efflux. With high hematocrit that gives faster cholesterol efflux, the efflux was increased directly by the cholesterol esterification. On the other hand, the LCAT reaction significantly reduced HDL-cholesterol influx. The LCAT reaction thus induces substantial net cholesterol efflux from erythrocytes through a nonspecific cholesterol exchange mechanism.

Human plasma apolipoprotein E (apo-E) strongly self-associates to form a stable tetramer in an aqueous solution at pH 7.4 containing 0.15 M NaCl. Tetramerized apo-E is abundant in alpha-helical conformation with an asymmetric molecular shape. Apo-E forms a stable monolayer at the air-water interface with a collapse pressure of 14 dynes/cm and with a limiting molecular area of 21 A2/amino acid. Under low surface pressure (less than 0.5 dyne/cm), it behaves as a monomer at the interface. It binds reversibly to the surface of phosphatidylcholine-coated triolein particles with a diameter of 26 nm from the aqueous phase in which most of the molecules are tetramerized. An apparent dissociation constant (Kd), 1.2 X 10(-6) M (monomeric molarity) or 40 mg/l, is substantially larger than those of the other apolipoproteins, while a binding saturation level (N), 0.8 amino acid/surface phospholipid, is equivalent to the N values of those proteins (Tajima, S., Yokoyama, S., and Yamamoto, A. (1983) J. Biol. Chem. 258, 10073-10082). Content of alpha-helix increases slightly when it is transferred from the aqueous phase to the lipid surface. The results are consistent with a model that amphiphilic alpha-helical conformation is responsible both for self-association and surface binding and suggest that apo-E may easily dissociate from the lipoprotein surface to form a self-associated soluble tetramer.

The allosteric effect of fructose 1,6-bisphosphate (Fru-1,6-P2) on L-lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from Thermus caldophilus GK24 was studied by means of 1H NMR analyses. The conformation of NAD+ as bound to the T. caldophilus enzyme was elucidated by analyses of the transferred nuclear Overhauser effects (TRNOE), in the presence and the absence of the allosteric effector, Fru-1,6-P2. Upon binding of Fru-1,6-P2 to the enzyme, the ribose ring of the adenosine moiety of NAD+ is converted from the C2'-endo form to the C3'-endo form. This C3'-endo form of the adenosine moiety is similar to that of NAD+ as bound to nonallosteric vertebrate enzymes. However, the anti conformation of the adenine-ribose bond of NAD+ as bound to the T. caldophilus enzyme is not affected by the binding of Fru-1,6-P2. In contrast, the syn conformation of the nicotinamide-ribose bond is converted to the anti form on the binding of Fru-1,6-P2, while the ribose ring remains in the C3'-endo form as found in the case of a nonallosteric enzyme. Such a conformational change of enzyme-bound NAD+ as found on TRNOE analysis is essentially involved in the allosteric regulation of the T. caldophilus enzyme by Fru-1,6-P2.

Cholesterol was poorly available to free apolipoprotein (apo)A-I-mediated cellular lipid efflux from cholesterol-loaded rat vascular smooth muscle cells generating cholesterol-poorer pre-beta-HDL particles than those generated from macrophages by the same reaction (Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). The factors known to induce transformation of the smooth muscle cells into a macrophage-like stage were used in order to modulate this reaction, such as human platelet-derived growth factor, macrophage colony-stimulating factor, and phorbol 12-myristate-13-acetate (PMA). When the cells were stimulated by PMA following the pretreatment with platelet-derived growth factor plus macrophage colony-stimulating factor, cholesterol efflux mediated by free apoA-I increased 3-fold without changing phospholipid efflux, resulting in generation of pre-beta-HDL particles more rich in cholesterol. This treatment had only a little or no effect on apparent cellular cholesterol efflux to HDL or lipid microemulsion, respectively. Overall cellular free cholesterol pool size was unaffected by the treatment, and probing by extracellular cholesterol oxidase did not detect gross change in the cellular surface cholesterol. This specific enrichment of cholesterol in the apoA-I-mediated cellular lipid efflux was reversed by protein kinase C inhibitors. Measurement of intracellular cholesterol esterification suggested that PMA induced translocation of intracellular cholesterol to a specific pool for apoA-I-mediated efflux, and a protein kinase C inhibitor reversed this effect.

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002-sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.

The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue. Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E. coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not). The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S. These results indicate that the zinc atom present in E. coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127. Molecular modeling of the N-terminal half of this enzyme, using the known structure of E. coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers.

Cerebellar granule neurons possess a non-inactivating K+ current, which controls resting membrane potentials and modulates the firing rate by means of muscarinic agonists. kcr1 was cloned from the cerebellar cDNA library by suppression cloning. KCR1 is a novel protein with 12 putative transmembrane domains and enhances the functional expression of the cerebellar non-inactivating K+ current in Xenopus oocytes. KCR1 also accelerates the activation of rat EAG K+ channels expressed in Xenopus oocytes or in COS-7 cells. Far-Western blotting revealed that KCR1 and EAG proteins interacted with each other by means of their C-terminal regions. These results suggest that KCR1 is the regulatory component of non-inactivating K+ channels.

On modification of arginine residues with 2,3-butanedione, the Thermus caldophilusL-lactate dehydrogenase is converted to an activated form that is independent of an allosteric effector, fructose 1,6-bisphosphate (Fru-1,6-P2). The conformation of NAD+bound to the modified enzyme in the absence of Fru-1,6-P2was investigated by means of proton NMR, analyzing the time dependence of the transferred nuclear Overhauser effect (TRNOE) and TRNOE action spectra. The inter-proton distances determined on TRNOE analysis indicated that both the nicotinamide riboside moiety and the adenosine moiety of NAD+were in the anti conformation, the ribose rings being in the C3′-endo form. This conformation was almost the same as that of NAD+bound to the native enzyme-Fru-1,6-P2complex, rather than that of NAD+ bound to the free native enzyme. These results suggest that the C3′-endo-antiform of the enzyme-bound NAD+is essential for the activation of the T. caldophilusL-lactate dehydrogenase.