共查询到20条相似文献,搜索用时 31 毫秒
1.
Modulation of isolation-induced fighting by N- and C-terminal analogs of substance P: evidence for multiple recognition sites 总被引:1,自引:0,他引:1
Substance P (SP) significantly reduced fighting in mice made aggressive by prolonged isolation. The N-terminal heptapeptide fragment SP (1-7) also reduced fighting. The C-terminal fragment SP(4-11) was without activity, while the shorter C-terminal fragment analog less than E-SP(7-11) significantly increased isolation-induced fighting. The aggression-enhancing effect of less than E-SP(7-11) was antagonized by naloxone, which by itself had no significant effect. The aggression-reducing effect of SP(1-11) was significantly enhanced by naloxone, while the effect of SP(1-7) was unchanged. These results demonstrate that a behavioral effect of SP may be duplicated by an N-terminal fragment of the SP molecule, and that peptide fragments or analogs of the N- and C-terminal portions of the SP molecule can exert opposing effects on a specific behavior. These findings represent a structure/activity relationship that is strikingly different from any previously described for SP. The differing effects of naloxone on N- and C-terminal fragment analogs suggest that these two effects may be mediated by different mechanisms. 相似文献
2.
Z Hrabec J Szkudlarek L Lachowicz 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1990,96(1):59-63
1. The effects of substance P and its fragments and analogue of a C-terminal fragment on cyclic AMP-dependent phosphorylation of synapsin I in synaptosomal membranes (SM) from cerebral cortex were investigated. 2. SP(I-II) and SP(1-4) at 10(-3) M caused a marked stimulation of synapsin I phosphorylation. 3. A C-terminal fragment of SP (SP6-11) had no effect on phosphorylation of synapsin 1. 4. Analogue of C-terminal fragment [(Tyr8)SP6-11] at 10(-3) M distinctly inhibits phosphorylation of synapsin I. 5. These data suggest that SPI-II and its C- and N-terminal fragments have a modulator function against the phosphorylation of some rat brain proteins. 相似文献
3.
Substance P (SP) injected intracerebroventricularly (ICV) into rabbits caused dose-related thermal analgesia with the maximum effect after 2 micrograms. The analgesia was measured by timing the withdrawal of the rabbit's ear from an infrared beam. Equimolar amounts of the related peptides physalaemin and eledoisin-related peptide also caused analgesia, but the SP N-terminal fragment (1-9) was inactive. This suggests that the analgesic message of SP resides within the C-terminal fragment. The analgesia caused by each peptide developed more rapidly but did not last as long as that after central injection of beta-endorphin. In separate experiments, 2 micrograms SP injected ICV increased blood pressure and decreased heart rate. The analgesic, bradycardic and pressor responses to central administration of SP were opposite to effects of peripherally administered SP, described previously. These results indicate that the effect induced by SP depends upon its specific neuroanatomical site of action. 相似文献
4.
Intrathecal injection of mice with substance P (SP) or its C-terminal fragments results in a behavioral syndrome characterized by reciprocal caudally directed biting and scratching. Repeated injection of SP, but not SP C-terminal fragments, results in a decrease in the intensity of, or desensitization to, these SP-induced behaviors. Peptidase inhibitors, phosphoramidon (PH), bacitracin (BAC), diprotin A (DPA) and angiotensin converting enzyme inhibitor (ACEI OR SQ20881), together with [3H]SP, were used to investigate the possible accumulation of tritiated N-terminal metabolites in the mouse spinal cord in vivo during the development of desensitization to SP. SP N-terminal metabolites in the spinal cord were quantified by reverse-phase HPLC. The magnitude of SP-induced desensitization correlated well (r = .95) with total SP N-terminal metabolites recovered from the spinal cords of the same mice studied in vivo. The magnitude of SP-induced desensitization was also found to be negatively correlated (r = .95) with total recovered intact [3H]SP. The rank order of potency of the peptidase inhibitors in decreasing the magnitude of SP-induced desensitization was BAC = PH much greater than ACEI greater than DPA. The order of potency for in vitro inhibition of SP metabolism using synaptic membrane-derived peptidases was BAC greater than PH much greater than ACEI. These results support the hypothesis that desensitization to SP-induced behaviors depends, at least in part, on the concentration of SP N-terminal metabolites in the spinal cord. 相似文献
5.
Anabolic-androgenic steroids affect the content of substance P and substance P(1-7) in the rat brain 总被引:1,自引:0,他引:1
The effects of intramuscular (i.m.) injections of nandrolone decanoate (15 mg/kg/day), an anabolic-androgenic steroid, on the levels of substance P (SP) and on its N-terminal fragment SP(1-7) were examined in the male rat brain by radioimmunoassay. The results demonstrated that the SP immunoreactivity in amygdala, hypothalamus, striatum, and periaqueductal gray was significantly enhanced, whereas the concentration of the N-terminal fragment SP(1-7) was enhanced in the nucleus accumbens and in periaqueductal gray. In the striatum the steroid induced a decrease in the content of SP(1-7). The relevance of these peptides in connection with anabolic-androgenic steroid-induced aggression is discussed. 相似文献
6.
We evaluated the chemotactic activity of beta-endorphin and beta-endorphin-related peptides on human monocytes. We tested beta-endorphin(1-31) and fragments (1-16), (1-17), (1-27) in which the N-terminal of the opioid is preserved, N-acetyl-beta-endorphin(1-31) and fragments (6-31) and (28-31) in which the C-terminal is preserved, and fragment (2-17) that lacks both the N- and C-terminal. The fragments in which the N- and C-terminal were preserved [with the exception of fragment (28-31)] showed a chemotactic effect, while the lack of both terminals deprived the peptides of any activity. Moreover, only the N-terminal-mediated effects were naloxone reversible, while the C-terminal effects were not. These results indicate that while the intact N-terminal is necessary for opioid like effects, both N- and C-terminal can mediate effects on the immune system, thus offering evidence for a nonopioid receptor-mediated effect of opioid peptides on the immune system. 相似文献
7.
Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1-7) (SPN) and the C-terminal analog [pGlu6]-SP(6-11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 micrograms/kg), SPN (3.3, 33, 167, 333 micrograms/kg) or SPC (2.7, 27, 134, 268 micrograms/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 micrograms/kg SP (37 nmol/kg) and 167 micrograms/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 micrograms/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 micrograms/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall. 相似文献
8.
Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (KD(1) less than 0.1 nM and KD(2) approximately 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (KD approximately 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD approximately 1 nM) or its N-terminal fragment galanin-(1-13) (KD approximately 1 microM), which constitutes the N-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (KD = 0.4 microM), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp2], in the galanin portion of the chimeric peptide or substitution of ([Phe7] or [Met11]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
The behavioral effects of the amino (N)-terminal fragment of substance P (SP(1-7)) on the marmoset (Callithrix penicillata) predator confrontation test of fear/anxiety were investigated. The test apparatus consisted of a figure-eight maze with three parallel arms interconnected at each extremity to a perpendicular arm. A taxidermized oncilla cat (Felis tigrina) was placed outside the maze facing one of its corners. Subjects were submitted to seven 30 min maze habituation trials (HTs), in the absence of the 'predator', and then to six 30 min treatment trials (TTs), in the presence of the 'predator', consisting of four doses of SP(1-7) (5, 50, 250 and 500 microg/kg; IP), saline and sham injection. SP(1-7) treatment reversed, in a dose-dependent way, the fear-induced avoidance behavior due to the predator's presence and increased the frequency of exploratory behaviors. Locomotor activity decreased during successive HTs, yet increased after all SP(1-7) treatments. These results indicate that systemic administration of SP(1-7) produces anxiolytic-like effects in marmosets tested in the predator confrontation model of fear/anxiety. 相似文献
10.
Use of substance P fragments to differentiate substance P receptors of different tissues 总被引:3,自引:0,他引:3
M F Piercey P J Dobry F J Einspahr L A Schroeder N Masiques 《Regulatory peptides》1982,3(5-6):337-349
The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively. 相似文献
11.
Fan Y Rafaeli A Moshitzky P Kubli E Choffat Y Applebaum SW 《Insect biochemistry and molecular biology》2000,30(8-9):805-812
Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations. 相似文献
12.
p67(PHOX), a cytosolic component of the NADPH oxidase complex, is phosphorylated during neutrophil activation by several agonists. The intracellular signaling pathways leading to its phosphorylation in neutrophils may involve a PKC-dependent pathway and a PKC-independent pathway. Here, we analyzed p67(PHOX) phosphorylation by ERK2 and p38MAPK. Both ERK2 and p38MAPK phosphorylated p67(PHOX) in vitro, with similar K(m) values (10 and 9 microM, respectively). Phosphopeptide mapping indicated that ERK2 and p38MAPK phosphorylate different subgroups of peptides. Using truncated forms of p67(PHOX), we found that the major phosphorylation target site of ERK2 was located in the N-terminal fragment (1-243), while the major phosphorylation target sites of p38MAPK were located in the C-terminal fragment (244-526). Furthermore, an additional peptide, which was not phosphorylated in the intact protein, appeared to be phosphorylated in the isolated C-terminal fragment (aa 244-526). This site may not thus be accessible in the intact protein. Indeed, incubation of the C-terminal fragment (244-526) with different N-terminal fragments (1-243, 1-210, or 1-199) containing the tetratricopeptide-rich region prevented phosphorylation of this C-terminal fragment. ERK1/2 and p38MAPK are also involved in p67(PHOX) phosphorylation in intact neutrophils. Indeed, PD98059 and SB203580, two selective inhibitors of MEK1/2 and p38MAPK, respectively, inhibited p67(PHOX) phosphorylation in fMLP- and PMA-stimulated neutrophils, with additive effects, thus suggesting that they also target different sites in vivo. Furthermore, the major peptides phosphorylated by ERK2 and p38MAPK in vitro were also phosphorylated in fMLP-stimulated neutrophils. Taken together, these results suggest not only that p67(PHOX) is phosphorylated by ERK2 and p38MAPK in vitro and in intact neutrophils on several selective sites but also that a C-terminal phosphorylation site may become accessible after a conformational change of the protein. 相似文献
13.
Botros M Hallberg M Johansson T Zhou Q Lindeberg G Frändberg PA Tömböly C Tóth G Le Grevès P Nyberg F 《Peptides》2006,27(4):753-759
Endomorphin-1 (EM-1) and endomorphin-2 (EM-2) represent two opioid active tetrapeptides with high affinity and selectivity for the mu-opioid (MOP) receptor. Both EM-1 and EM-2 exhibit strong inhibition of pain signals in the central nervous system (CNS). In contrast to these compounds, the undecapeptide substance P (SP) facilitates pain influx in the CNS. SP has been implicated in a number of functions in the central nervous system, including pain processing and reward. Its aminoterminal fragment SP1-7 has been shown to modulate several actions of SP in the CNS, the nociceptive effect included. Although the actions of SP1-7 have been known for long no specific receptor for the SP fragment has yet been cloned. In this study, we demonstrate the presence of specific binding sites for the heptapeptide in the rat spinal cord. The binding affinity for unlabeled SP1-7 to the specific sites for the labeled heptapeptide highly exceeded those of SP and other C- or N-terminal fragments thereof. The NK-1, NK-2 and NK-3 receptor ligands [Sar9, Met(O2)11]SP, R396 and senktide, respectively, showed no or negligible binding. Moreover, both EM-1 and EM-2 were found to interact with SP1-7 binding. However, a significant difference in binding affinity between the two opioid active tetrapeptides was observed. As recorded from replacement curves the affinity of EM-2 was 10 times weaker than that for SP1-7 but about 100 times higher than that of EM-1. Among other Tyr-Pro-containing peptides Tyr-MIF-1 but not Tyr-W-MIF-1 exhibited affinity of similar potency as EM-2. These results strengthen the previously observed differences between EM-1 and EM-2 in various functional studies. Moreover, using a cell line (C6) expressing the MOP receptor it was shown that the labeled SP1-7 did not interact with binding to this receptor and no functional response was seen for the SP heptapeptide on the MOP receptor by means of stimulation in the GTPgammaS assay. This suggests that the identified SP1-7 binding sites, with high affinity also for EM-2, are not identical to the MOP receptor and apparently not to any of the known tachykinin receptors. 相似文献
14.
G Lindner 《Zeitschrift für mikroskopisch-anatomische Forschung》1984,98(1):107-118
The effect of peptides (SP1-2, SP3-4 and SP5-11) of substance P (SP1-11) on the morphology of the areas of growth of explants of the ganglion trigeminale from chick embryos after incubation in Maximow chambers was observed. N- and C-terminal sequences effected the growth of cultures differently. In dipeptide-treated (= N-terminal sequences) cultures the areas of growth increased. In heptapeptide-treated cultures (= C-terminal sequence SP5-11) the areas of growth decreased. Only the dipeptide SP3-4 effects a mitogenic effect on nonneuronal cells in short time tests. The C-terminal sequence SP5-11 stimulates neither the growth of nerve fibres nor the proliferation of cells. Finally the importance of this in-vitro-tests in relation to the in-vivo-situation is discussed. 相似文献
15.
Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane 总被引:18,自引:0,他引:18
A Davies K Meeran M T Cairns S A Baldwin 《The Journal of biological chemistry》1987,262(19):9347-9352
Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots. 相似文献
16.
The N-terminal metabolite of the undecapeptide substance P (SP), substance P1-7 (SP1-7), is known to modulate nociception in the central nervous system (CNS) and often has opposite effects from SP. This study investigated the ability of SP(1-7) to modulate the vasodilatation response to SP in anaesthetized rats under different injury conditions using a blister model of inflammation on the hind footpad. The results indicated that SP1-7 inhibited the vascular response to SP in a dose-dependent manner. The putative antagonists naloxone and D-Pro2-D-Phe7-SP1-7 (D-SP1-7) reversed the effect of SP1-7. D-SP1-7 improved the responsiveness to SP under chronic nerve injury, which suggests a role for endogenous SP1-7 in this model. SP1-7 did not inhibit the response to electrical stimulation of the sciatic nerve, which indicates that the heptapeptide interacts at a post-terminal binding site. The current results suggest that SP1-7 may have inhibitory properties in inflammation, analogous to its antinociceptive role in the central nervous system. 相似文献
17.
To help define the immunological epitopes on human growth hormone (hGH), interaction of fragments of the hormone with 7 monoclonal antibodies (McAbs) was studied. Plasmin-digested hGH, containing two peptides (hGH1-134 and hGH141-191) joined by a disulphide bond, bound to each McAb with affinity similar to that of intact hGH. The purified C-terminal fragment, hGH141-191, showed low affinity for each McAb. The N-terminal fragment, hGH1-134, bound with quite high affinity to 2 McAbs (EB1 and EB3) but not to the other 5. We conclude that residues 1-134 of hGH contain the epitope to which McAbs EB1 and EB3 bind. 相似文献
18.
M Hiroyoshi K Kaibuchi S Kawamura Y Hata Y Takai 《The Journal of biological chemistry》1991,266(5):2962-2969
Limited proteolysis with trypsin of smg p21B, a ras p21-like small GTP-binding protein having the same putative effector domain as ras p21s, produced the N-terminal fragment and the C-terminal tail of Lys-Lys-Ser-Ser-geranylgeranyl-Cys methyl ester. The Mr values of the intact smg p21B, the N-terminal fragment, and the C-terminal tail were estimated to be about 22,000, 20,500, and less than 1,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the GDP- and GTP-bound forms of the intact smg p21B bound to various membranes and phosphatidylserine-linked Affi-Gel. However, both the GDP- and GTP-bound forms of the N-terminal fragment failed to bind to membranes and phosphatidylserine-linked Affi-Gel. In contrast, the C-terminal tail bound to membranes and phosphatidylserine-linked Affi-Gel. The N-terminal fragment contained a GDP/GTP-binding and GTPase domain and exhibited these two activities, but the C-terminal tail did not show any such activity. A GTPase-activating protein for smg p21 stimulated the GTPase activity of both the intact smg p21B and the N-terminal fragment. In contrast, a GDP/GTP exchange protein for smg p21, named GDP dissociation stimulator, stimulated the GDP/GTP exchange reaction of the intact smg p21B but not that of the N-terminal fragment. These results indicate 1) that smg p21B is composed of at least two functionally different domains, the N-terminal GDP/GTP-binding and GTPase domain and the C-terminal membrane-binding domain, 2) that smg p21B binds to membranes through its C-terminal hydrophobic and basic domain, and 3) that this C-terminal domain is also essential for the smg p21 GDP dissociation stimulator action but not for the smg p21 GTPase-activating protein action. 相似文献
19.
Nam Sang Cheung Per Karlsson Jian-Xin Wang Michael Bienert† Peter Oehme† Bruce G. Livett 《Journal of neurochemistry》1994,62(6):2246-2253
Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr12-NH2, 65% by (Ala3)SP-NH2, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr12-NH2 > (Ala3)SP-NH2 > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala3)SP-NH2 protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr12-NH2 at 3 × 10-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH2, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently. 相似文献
20.
Probing the limits of the DNA breakage-reunion domain of the Escherichia coli DNA gyrase A protein. 总被引:7,自引:0,他引:7
In a previous report (Reece, R. J., and Maxwell, A. (1989) J. Biol. Chem. 264, 19648-19653) we showed that treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two stable fragments. The N-terminal 64-kDa fragment supports DNA supercoiling, while the C-terminal 33-kDa fragment shows no enzymic activity. We proposed that the 64-kDa fragment represents the DNA breakage-reunion domain of the A protein. We have now engineered the gyrA gene such that the 64-kDa protein is generated as a gene product. The properties of this protein confirm the findings of the experiments with the 64-kDa tryptic fragment. We have also generated a series of deletions of the gyrA gene such that C-terminal and N-terminal truncated versions of the A protein are produced. The smallest of the N-terminal fragments found to be able to carry out the DNA breakage-reunion reaction is GyrA(1-523). The cleavage reaction mediated by this protein occurs with equal efficacy as that performed by the intact GyrA protein. Deletion of the N-terminal 6 amino acids from either the A protein or these deletion derivatives has no effect on enzymic activity, while deletion of the N-terminal 69 amino acids completely abolishes the DNA breakage-reunion reaction. Therefore the smallest GyrA protein we have found that will perform DNA breakage and reunion is GyrA(7-523). A model is proposed for the domain organization of the gyrase A protein. 相似文献