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PubMed2005

The Saccharomyces cerevisiae ESU1 gene, which is responsible for enhancement of termination suppression, corresponds to the 3'-terminal half of GAL11.

A DNA fragment enhancing efficiency of [PSI+]-dependent termination suppressor, sup111, was isolated from a genomic library of Saccharomyces cerevisiae and its function was attributed to an ORF of 1272 bp. This ORF, designated ESU1 (enhancer of termination suppression), corresponded to the 3'-terminal portion of GAL11. Contrasting to ESU1, GAL11 lowered the suppression efficiency of [PSI+] sup111. ESU1 possesses a TATA-like sequence of its own and three ATG codons following it within a distance of about 70 bp and all in the same reading frame as GAL11. A 52.7 kDa protein corresponding in size to the predicted Esu1 protein is detected by western blot analysis using anti-Gal11 antiserum. We therefore conclude that ESU1 is the gene that encodes a polypeptide corresponding to the C-terminal 424 amino acids of Gal11. It was further found that ESU1 increases the level of GAL11 mRNA and probably also of its own mRNA. Moreover, ESU1 increased the cellular level of mRNA transcribed from the leu2-1(UAA) mutant gene, while GAL11 did not. Based on these findings, we propose the following scheme for the events taking place in the [PSI+] sup111 cell that is transformed with an ESU1-bearing plasmid: (a) ESU1 stimulates transcription of leu2-1; (b) leu2-1 mRNA is not effectively degraded because of the possession of sup111, which belongs to the upf group; (c) [PSI+] causes increased mis-termination due to depletion of eRF3; (d) functional Leu2 product is made using leu2-1 mRNA; and (d) suppression of leu2-1 is eventually accomplished.

Galactose↗
PubMed1998

Hard alpha-keratin intermediate filament chains: substructure of the N- and C-terminal domains and the predicted structure and function of the C-terminal domains of type I and type II chains.

The quantity of sequence data now available for both Type I and Type II hard alpha-keratin IF proteins makes it possible to analyze their N- and C-terminal domains and ascertain features of likely structural and/or functional importance. The N-terminal domains of both chain types can be divided into acidic (NA) and basic (NB) subdomains, where NA is 29 and 34 residues long, respectively, for Type I and II chains and is located immediately adjacent to the end of the rod domain. NB constitutes the remainder of the N-terminal domain and is about 27 and 70 residues long for the two chain types, respectively. The glycine residue contents, however, are high in NA(I) and NB(II), but low in NA(II) and NB(I). Subdomain NB(II) contains four consecutive nonapeptide quasirepeats of the form GGGFGYRSX. The C-terminal domain of Type I chains, termed C(I), is characterized by a PCX motif repeated 10 times, 7 of them contiguously. From an analysis of the conformation of like peptides from crystal structures it has been shown that this region will probably adopt a polyproline II left-handed helical structure with three residues per turn. In contrast, the C-terminal domain of Type II hard alpha-keratin chains (known as C(II)) contains a periodic distribution of hydrophobicities that, together with other predictive techniques, allow its conformation (a twisted four-stranded antiparallel beta-sheet) to be predicted with some degree of confidence. In addition, it is possible to suggest two partners with which this domain will interact. The first is with segment L12 in the rod domain and the second is with another C(II) domain in an antiparallel neighboring molecule. The latter possibility appears most likely. In either case the aggregation would likely serve to stabilize the molecular assembly through the interaction of two beta-sheets via their apolar faces and, in so doing, would position a number of cysteine residues in external positions that would allow them to form a number of covalent disulfide bonds with other molecules.

Amino Acid Sequence↗
PubMed1978

Radioautographic study of 5-hydroxytryptamine-containing nerve terminals in central ganglia of Planorbis corneus: comparison with other species and characteristics of the serotoninergic nerve terminal.

Central ganglia from Planorbis corneus were incubated with [3H]5-hydroxytryptamine and [3H]-leucine. After fixation, sections were examined by light and electron microscopic autoradiography. With [3H]5-hydroxytryptamine there was a high level of uptake into a small number of axons and terminal processes in the neuropil. The terminal processes contained granular vesicles (diameter 50--120 nm), some of which possessed eccentrically placed cores, and agranular vesicles (mean diameter 60 nm). No membrane synaptic specializations were observed. With [3H]leucine there was a general distribution of radioactivity throughout the ganglia. The vesicular inclusions and non-specialized nature of the labelled terminal processes were very similar to presumed 5-hydroxytryptamine-containing terminals in other invertebrates and in the mammalian brain. It appears that several anatomical features of serotoninergic nerve terminals are common in all animals studied, although there is no specific characteristic which allows positive identification.

Animals↗
PubMed1985

Production and characterization of N-terminally and C-terminally directed monoclonal antibodies against pancreatic glucagon.

Hybridoma technology has been successfully applied to the production of monoclonal antibodies against a variety of small soluble peptides. We report herein for the first time on the development of monoclonal antibodies to pancreatic glucagon. Twenty-three stable positive hybridomas were detected by radioimmunoassay from five separate fusions and cloned by the limiting dilution method. Four selected monoclonal antibodies were all of the IgG 2a subclass type kappa and bound to protein A. One monoclonal antibody (23.8B6) was shown to be directed toward the C-terminal region and another (23.6B4) toward the N-terminal to central region of the glucagon molecule. These antibodies did not cross-react with any of the other peptides tested. Two further monoclonal antibodies (23.4A1, 22.3A6) reacted with the C-terminal third of the glucagon molecule and showed a cross-reaction with the structurally related gastric inhibitory polypeptide of 0.7% and 9.1%, respectively. All but the C-terminal monoclonal antibody 23.8B6 showed a marked cross-reaction with ileal extracts. The N-terminally directed monoclonal antibody 23.6B4 was of sufficient avidity for use in the radioimmunoassay of pancreatic glucagon and gut glucagon-like immunoreactivity in tissue extracts, being sensitive to changes of pancreatic glucagon of 2.0 fmol/tube at a final titer of culture supernatant of 1:1.4 X 10(5). In gel permeation chromatography of intestinal extracts, two major peaks were detectable (Kav 0.27 and 0.54). The present findings show that monoclonal antibodies provide sensitive tools for detecting pancreatic glucagon and gut glucagon-like immunoreactivity. They will be valuable immunoreactants for the development of immunoradiometric assays as well as for large-scale immunoaffinity purification of gut glucagon-like immunoreactivity.

Animals↗
PubMed1982

Bleomycin pulmonary toxicity: production of fibrosis by bithiazole-terminal amine and terminal amine moieties of bleomycin A2.

We have previously demonstrated that endotracheal administration of the terminal amines of several bleomycins, when administered as the free amines, produce pulmonary fibrosis of severity comparable to the intact drug. In the present report, bleomycin A2 and its sulfonium ion-containing terminal substituents, with and without the bithiazole rings, were administered to mice endotracheally. The incidence and severity of epithelial metaplasia was greater with the intact drug in comparison to the terminal substituents. In contrast, the terminal substituents and intact drug produced similar degrees of fibrosis. These results underscore the importance of the variable bleomycin terminal substituents in the pathogenesis of pulmonary fibrosis.

Animals↗
PubMed1991

Immunoreactive N-terminal fragment of proatrial natriuretic peptide, ANP (1-98), in plasma of healthy dogs and dogs with impaired volume regulation: a comparison with the C-terminal ANP (99-126).

Atrial natriuretic peptide (ANP) is stored in atrial myocytes as a 126 amino acid precursor molecule (ANP 1-126) and is cleaved during its release into circulation into the biologically active C-terminal ANP (99-126) and the N-terminal counterpart, ANP (1-98). While interest has focused on ANP (99-126) under physiological and pathophysiological conditions, data for the cosecreted N-terminal sequence, ANP (1-98) are generally missing. Plasma levels of the N-terminal immunoreactive peptide (N-ANP [1-98]) were measured in normal dogs, and in dogs with impaired volume regulation (congestive heart failure; chronic renal failure or Cushing's syndrome and compared with those of C-ANP (99-126). The N-ANP (1-98) concentration was 593.1 +/- 81.1 fmol ml-1 in normal subjects, which is about 60-fold higher than the C-ANP (99-126) (10.8 +/- 2.6 fmol ml-1). In patients suffering from chronic renal failure ANP (1-98) was increased to 1582 +/- 196 fmol ml-1, and in dogs with congestive heart failure to 1612 +/- 244 fmol ml-1. In contrast, Cushing's syndrome dogs showed decreased N-ANP (1-98) concentrations (351 +/- 65.9 fmol ml-1). There was a positive correlation between plasma levels of N-ANP (1-98) and C-ANP (99-126) levels (correlation coefficients: normal: r = 0.78; congestive heart failure: r = 0.76; chronic renal failure: r = 0.86; Cushing's syndrome: r = 0.57). High pressure liquid chromatographic analysis of dog plasma showed one major peak of N-terminal immunoreactivity corresponding to ANP (1-98).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Falciparum malaria parasitized erythrocytes bind to a carboxy-terminal thrombospondin fragment and not the amino-terminal heparin-binding region.

We investigated Plasmodium falciparum parasitized erythrocyte binding to proteolytic fragments of thrombospondin and the effects of anti-thrombospondin monoclonal antibodies on this binding. Purified human platelet thrombospondin was cleaved by trypsin, chymotrypsin or thrombin. Fragments were separated by heparin-agarose affinity chromatography, removing the amino-terminal heparin-binding region. Trypsin at 5.0 micrograms ml-1 of thrombospondin cleaved thrombospondin to reduced 140 and 120 kDa fragments plus a reduced 25-kDa heparin-binding fragment. Infected erythrocytes bound to intact thrombospondin (3420 +/- 460 infected erythrocytes mm-2) and the carboxy-terminal fragment, yielding 120-140-kDa fragments on sulfhydryl reduction, but not to the 25-kDa fragment (144 +/- 104 infected erythrocytes mm-2 (mean +/- s.d., N = 4). Similar results were obtained with chymotrypsin and thrombin cleavage. When the anti-thrombospondin monoclonal antibody MA-I was added to immobilized thrombospondin prior to infected erythrocytes, adherence was inhibited by 99%. At the same concentration, MA-I inhibited adherence to C32 melanoma cells by only 35%. MA-I binds to a calcium-dependent structure at the C-terminal globular region of thrombospondin. Monoclonal antibody MA-II inhibited adherence to thrombospondin by 46%, while MA-III had no effect. These antibodies bind to the N-terminal globular region which includes the heparin-binding site and the segment connecting the two globular regions, respectively. The site(s) for infected erythrocyte binding on thrombospondin reside in the large, 140- or 120-kDa, proteolytic cleavage fragments, and not in the N-terminal heparin-binding region.

Animals↗
PubMed2005

Role of C-terminal regions of the C-terminal fragment of Clostridium perfringens enterotoxin in its interaction with claudin-4.

Claudin family proteins, which contain 4 transmembrane domains, play a pivotal role in the barrier function of tight junctions (TJs) in epithelial sheets. We previously found that a modulator of claudin-4, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), is a potent enhancer of jejunal drug absorption in rats. But the effects of C-CPE on the barrier function of TJs have never been fully understood. In the present study, we investigated the effects of C-CPE on the barrier function of TJs in Caco-2 monolayer and characterized the functional domain of C-CPE that is responsible for interaction with claudin-4. To evaluate the effects of C-CPE on the barrier function of TJs, we measured transepithelial electric resistance (TER) in Caco-2 monolayer cells seeded onto polycarbonate filters. Treatment of Caco-2 cells with C-CPE resulted in a decrease in TER. But, deletion of the 30 C-terminal amino acids of C-CPE, which is the putative binding domain for claudin, attenuated the decrease in TER values. Moreover, ablation of the 16 C-terminal amino acids of C-CPE also resulted in attenuation of the decrease in TER values. The C-terminal-deleted C-CPEs did not interact with claudin-4 or the extracellular domain 2 of claudin-4, which is the C-CPE binding site. These results suggest that the 16 C-terminal amino acids of C-CPE are responsible for the interaction of C-CPE and claudin-4 following the disruption of TJ barrier function.

Caco-2 Cells↗
PubMed1997

Glycine-immunoreactive terminals in the rat trigeminal motor nucleus: light- and electron-microscopic analysis of their relationships with motoneurones and with GABA-immunoreactive terminals.

Post-embedding immunolabelling methods were applied to semi-thin and ultrathin resin sections to examine the relationships between glycine- and gamma-aminobutyric acid (GABA)-immunoreactive terminals on trigeminal motoneurones, which were identified by the retrograde transport of horseradish peroxidase injected into the jaw-closer muscles. Serial sections were cut through boutons and alternate sections were incubated with antibodies to glycine and GABA. Light-microscopic analysis of semi-thin sections revealed a similar pattern of glycine and GABA-immunoreactive boutons along the motoneurone soma and proximal dendrites, and of immunoreactive cell bodies in the parvocellular reticular and peritrigeminal areas surrounding the motor nucleus. Immunoreactive synaptic terminals on motoneurones were identified on serial ultrathin sections at electron-microscopic level using a quantitative immunogold method. Three populations of immunolabelled boutons were recognized: boutons immunoreactive for glycine alone (32%), boutons immunoreactive for GABA alone (22%), and boutons showing co-existence of glycine and GABA immunoreactivities (46%). Terminals which were immunoreactive for glycine only contained a higher proportion of flattened synaptic vesicles than those which were immunoreactive for GABA only, which contained predominantly spherical vesicles. Terminals which exhibited both immunoreactivities contained a mixture of vesicle types. All three classes of terminal formed axo-dendritic and axo-somatic contacts onto retrogradely labelled motoneurones. A relatively high proportion (25%) of boutons that were immunoreactive for both transmitters formed synapses on somatic spines. However, only GABA-immunoreactive boutons formed the presynaptic elements at axo-axonic contacts: none of these were found to contain glycine immunoreactivity. These data provide ultrastructural evidence for the role of glycine and GABA as inhibitory neurotransmitters at synapses onto jaw-closer motoneurones, but suggest that presynaptic control of transmission at excitatory (glutamatergic) synapses on motoneurones involves GABAergic, but not glycinergic inhibition.

Animals↗
PubMed1997

The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein.

In order to localize the active site of the vitamin K-dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-terminal and hydrophilic carboxy-terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino-terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino-terminal fragment is seriously underestimated by SDS-PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy-terminal hydrophilic domain.

Amino Acid Sequence↗
PubMed2001

Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein.

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K(i) of 1.04, 1.34 and 0.52 microM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K(i) of 332 and 56 microM, respectively) whilst the Hairy peptide bound much more strongly (K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.

Adenovirus E1A Proteins↗
PubMed1997

Molecular cloning and N-terminal analysis of bovine cystatin C. Identification of a full-length N-terminal region.

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Amino Acid Sequence↗
PubMed1999

Anxiogenic effects of substance P and its 7-11 C terminal, but not the 1-7 N terminal, injected into the dorsal periaqueductal gray.

The dorsal periaqueductal gray matter (DPAG) is one of the main output regions of the brainstem for the expression of defense reaction. Recent findings implicating neurokinins in the expression of fear or anxiety-like behaviors, have stimulated interest in the participation of these neuropeptides in the generation of aversive states in the dorsal periaqueductal gray matter. Analyses of traditional measures of the behavior of rats submitted to the elevated plus-maze test in this laboratory have shown that microinjections of substance P (SP) into the DPAG produce anxiogenic-like effects. The present study employs an ethological analysis of the behavior of animals in this test to investigate the involvement of substance P (SP) and its C- and N- fragments (7-11 and 1-7) in the expression of the different aspects of fear upon injection into the DPAG. To this end, rats were implanted with a cannula in the DPAG and injected one week later with 35 and 70 pmol of either substance P, or C- or N- SP fragments and tested immediately afterwards in the elevated plus-maze. The results show that SP and its C terminal fragment, produced increases in scanning, stretched attend posture, head dipping and flat-back approach, whereas the fragment N terminal produced only an increase in rearing. Therefore, the effects of SP and its C terminal fragment were associated to risk assessment behavior, whereas those of N terminal fragment were related to vertical exploratory activity. The results indicate that SP produces anxiogenic effects through activation of neural substrates of aversion in the DPAG and that this effect is probably related to its C terminal fragment.

Animals↗
PubMed1993

A radioimmunoassay for the N-terminal propeptide of rat procollagen type III. Application to the study of the uptake of the N-terminal propeptide of procollagen type III in isolated perfused rat liver.

Antibodies against a synthetic peptide representing the 14 C-terminal amino acids of the N-terminal propeptide of rat and bovine procollagen type III were raised in rabbits and used to develop a radioimmunoassay. N-Terminal propeptide of procollagen type III, purified from calf skin, served as standard and tracer material. The IC50 of the standard inhibition curve was 2.1 micrograms/l, the lower limit of detection about 0.4 microgram/l, interassay variation was 8.5% and the intraassay variation 6.6% in typical experiments. Three peaks of antigenicity were detected in rat serum after gel chromatography. One peak coeluted with purified N-terminal propeptide of procollagen type III, one peak contained material approximately twice this size, and one peak eluted close to the void volume of the column. The antigen concentration in rat serum decreased in an age dependent manner. Rat, bovine, sheep and minipig serum antigen was sufficiently crossreactive to allow the application of the assay to these species, whereas human, goat and guinea pig samples were not. The degradation product Col 1, causing non-parallel inhibition in commercially available assays for human samples was not recognized since it does not contain the epitope represented by the synthetic peptide. The assay was used to study the half-life of bovine and endogenous N-terminal propeptide of procollagen type III in isolated perfused rat liver. [125I]-labeled antigen was cleared rapidly from the perfusate (t1/2 less than 5 min). The bovine antigen was removed from the perfusate with a half-life of 15 +/- 4 min. Endogenous propeptide was perfused for 120 min with little change in concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

N-terminal domain of Avena phytochrome: interactions with sodium dodecyl sulfate micelles and N-terminal chain truncated phytochrome.

Phytochrome is the ubiquitous red light photoreceptor present in plants. Properties of the 6-kDa end terminal region of phytochrome A (PHYA from etiolated Avena) have been investigated by the use of synthetic polypeptide fragments corresponding to that region. This region of the phytochrome A protein has been viewed as a possible functional site due to the large differences in the sequence's conformation and exposure between the Pr (red light-absorbing form) and Pfr (far-red light-absorbing, gene-regulating form) species of phytochrome A. Hydrophobic moment calculations reveal amphiphilic helical potential in this section of the protein, consistent with the folding of the N-terminal region onto a hydrophobic chromophore/chromophore pocket. A large N-terminal synthetic peptide also demonstrated helical folding in the presence of SDS micelles. This experimental evidence indicates that the N-terminal alpha-helical folding upon conversion of the regulatorily inactive Pr to the active Pfr form of phytochrome A is likely driven at least in part by amphiphilic helix stabilization. Further, the large synthetic peptide was spectrally demonstrated to interact with phytochrome A lacking the N-terminal region. The formation of this nativelike complex may provide us with a tool for both biophysical and physiological studies on the mechanism of phytochrome A signal transduction.

Edible Grain↗
PubMed1985

Aliphatic semisynthetic variants of the amino-terminal residue of sperm whale myoglobin: enrichment with 13C and determination and interpretation of terminal pK values.

The synthesis of a series of myoglobins substituted in the amino-terminal residue to provide variation in the aliphatic nature of the side chain and enrichment in 13C was accomplished by semisynthetic methods. The replacements for valine, the native first residue, included 13C-enriched glycine, alanine, valine, leucine, and isoleucine. The products were extensively characterized and found to be virtually indistinguishable by most physical methods. 13C NMR spectroscopy showed significant differences in the amino-terminal pK value, ranging from 7.72 for [Gly1]myoglobin to 7.15 for [Leu1]myoglobin. Consideration of the electrostatic effects of the charge matrix indicated a balance of interactions at this site not significantly altered by these variations in the side chain. By examination of the crystal structure, consideration of earlier work regarding the interactions of the side chain of Leu-2, and data regarding the motions of the terminal residue, it was concluded that the interaction of the side chain of the first residue with the hydrophobic cluster formed primarily by close contact of invariant residues Leu-2 and Leu-137 was the primary cause for the reduction in terminal pK values seen for the larger aliphatics. By restricting the freedom of the residue, this interaction limits the available hydration volume and consequently favors the unprotonated form of the amine. The concurrent observation of both functional elements in the series of alpha-amino-terminal residues brings out the interrelated consequences for the two categories of solvent interactions controlling structural and functional properties in a graded way.

Amino Acid Sequence↗
PubMed2007

3'-Azido-3'-deoxythymidine-(5')-tetraphospho-(5')-adenosine, the product of ATP-mediated excision of chain-terminating AZTMP, is a potent chain-terminating substrate for HIV-1 reverse transcriptase.

The resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) involves phosphorolytic excision of chain-terminating AZT-5'-monophosphate (AZTMP). Both pyrophosphate (PPi) and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. PPi-mediated excision produces AZT-5'-triphosphate (AZTTP), which could be immediately re-used as a substrate for viral DNA chain termination. In contrast, ATP-mediated excision produces the novel compound AZT-(5')-tetraphospho-(5')-adenosine (AZTp4A). Since little is known of the interaction of AZTp4A with HIV-1 RT, we carried out kinetic and molecular modeling studies to probe this. AZTp4A was found to be a potent inhibitor of HIV-1 RT-catalyzed DNA synthesis and of both ATP- and PPi-mediated AZTMP excision. AZTp4A is in fact an excellent chain-terminating substrate for AZT-resistant RT-catalyzed DNA synthesis, better than AZTTP (k(pol)/Kd = 6.2 and 11.9 for AZTTP and AZTp4A, respectively). The affinity of AZT-resistant HIV-1 RT for AZTp4A is at least 30,000-fold greater than that for the excision substrate ATP and approximately 10-fold greater than that for AZTTP. Dissociation of newly formed AZTp4A from RT may therefore provide a significant rate-limiting step for continued HIV-1 DNA synthesis. Our studies show that the products of PPi- and ATP-mediated excision of chain-terminating AZTMP (AZTTP and AZTp4A, respectively) are both potent chain-terminating substrates for HIV-1 RT, suggesting that there is no obvious benefit to HIV using ATP instead of PPi as the excision substrate.

Adenosine Triphosphate↗
PubMed1997

C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases.

The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.

Amino Acid Sequence↗